Engineered yeast as a method for bioremediation

ABSTRACT

Metal bioremediation and metal mining strategies can include compositions and methods.

CLAIM OF PRIORITY

This application claims the benefit of prior U.S. ProvisionalApplication No. 62/453,609 filed on Feb. 2, 2017, which is incorporatedby reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 12, 2018, isnamed 14952_0550_SL.txt and is 10,600 bytes in size.

TECHNICAL FIELD

This invention relates to metal bioremediation and metal mining.

BACKGROUND

Many heavy metals and organic compounds persistently contaminate publicwaters due to manufacturing processes, agricultural waste, and/or fromcorroding pipes and infrastructure. Examples such as lead, chromium, andcopper leakage can cause long term physiological damage, and eventualdeterioration of the respiratory organs and skeletal system. Seewww.epa.gov/airtoxics/hlthaf/chromium.html, which is incorporated byreference in its entirety. Chronic exposure, in most cases due to lackof water sanitation and monitoring, can cause long term health problemssuch as cancer and birth-related defects. Seewww.epa.gov/airtoxics/hlthef/hapglossaryrev.htm, which is incorporatedby reference in its entirety.

Similarly, there are a variety of human-made and artificially derivedorganic wastes that pervade the public's water system that both harm thepublic and damage the environment. By-products and runoff fromindustrial sites such as trichloroethylene (TCE) and other compoundshave been shown to affect the central nervous system, promote heartfailure, and even cause cancer. See Kjellstrand, P., el al.,“Irreversible effects of trichloroethylene exposure on the centralnervous system.” Scandinavian journal of work, environment & health(1980): 40-47, Watson, Rebecca E., et al.“Trichloroethylene-contaminated drinking water and congenital heartdefects: a critical analysis of the literature.” Reproductive Toxicology21.2 (2006): 117-147, and Wartenberg, Daniel, Daniel Reyner, and CherylSiegel Scott. “Trichloroethylene and cancer: epidemiologic evidence.”Environmental health perspectives108.Suppl 2 (2000): 161, each of whichis incorporated by reference in its entirety.

Similarly, there are human-made reservoirs of waste caused by mining anddrilling. For example, the oil sands, mainly centered in regions ofCanada around the Athabasca watershed, have made a huge environmentalimpact due to the massive drilling & mining in the area. Most of theindustrial work in the area has released toxic pollutants such asarsenic, chromium, lead, mixtures of reactive hydrocarbons, andirremediable tar. See Kelly, Erin N., et al., “Oil sands developmentcontributes elements toxic at low concentrations to the Athabasca Riverand its tributaries.” Proceedings of the National Academy of Sciences107.37 (2010): 16178-16183, which is incorporated by reference in itsentirety.

SUMMARY

In one aspect, a composition for remediating a metal to treat water caninclude a cell and a first oligomer of a metal binding protein expressedon a surface of the cell via a linker, where the linker can be tetheredto the first oligomer of the metal binding protein and to a surface ofthe cell, the metal binding protein has specificity for a metal, and thefirst oligomer of the metal binding protein expressed on the surface ofthe cell is capable of aggregating with a second oligomer of the metalbinding protein in the water upon binding a metal.

In certain embodiments, the cell can be yeast.

In certain embodiments, the linker can be a monomer of the metal bindingprotein.

In certain embodiments, the second oligomer of the metal binding proteincan be secreted from the cell.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the metal can be nickel, iron, copper, cobalt,lead, cadmium or mercury.

In certain embodiments, the metal binding protein can be glutaminesynthetase.

In certain embodiments, the metal binding protein can be fused with ahigh affinity protein including a metal binding domain, wherein themetal binding domain has specificity for the metal.

In certain embodiments, the high affinity protein can be a plantmetallothionein.

In certain embodiments, the metal binding protein can be expressed in ayeast host strain.

In another aspect, a method of remediating a metal to treat water caninclude preparing a composition comprising a cell and a first oligomerof a metal binding protein expressed on a surface of the cell via alinker, where the linker can be tethered to the first oligomer of themetal binding protein and to a surface of the cell, the metal bindingprotein has specificity for a metal, and the first oligomer of the metalbinding protein expressed on the surface of the cell is capable ofaggregating with a second oligomer of the metal binding protein in thewater upon binding a metal, and contacting water with the composition.

In certain embodiments, the method can further include adding the secondoligomer of the metal binding protein in the water.

In certain embodiments, the cell can be yeast.

In certain embodiments, the linker can be a monomer of the metal bindingprotein.

In certain embodiments, the second oligomer of the metal binding proteincan be secreted from the cell.

In certain embodiments, the metal can be a divalent metal

In certain embodiments, the metal can be nickel, iron, copper, cobalt,lead, cadmium or mercury.

In certain embodiments, the metal binding protein can be glutaminesynthetase.

In certain embodiments, the metal binding protein can be fused with ahigh affinity protein including a metal binding domain, wherein themetal binding domain has specificity for the metal.

In certain embodiments, the high affinity protein can be a plantmetallothionein.

In certain embodiments, the second oligomer of the metal binding proteincan be secreted from the cell via a signal peptide.

In certain embodiments, the signal peptide can be S. cerevisiae'sα-mating-factor.

In certain embodiments, the signal peptide can be AGA1/2 or EXG1.

In certain embodiments, the method can be performed at 20° C. or lowertemperature.

In certain embodiments, the metal binding protein can be expressed in ayeast host strain.

In certain embodiments, the yeast host strain can be Pichia pastoris.

In another aspect, a method of making a composition for remediating ametal to treat water can include selecting a cell, and expressing ametal binding protein on a surface of the cell, and tethering a firstoligomer of a metal binding protein expressed on a surface of the cellvia a linker, where the linker is tethered to the first oligomer of themetal binding protein and to a surface of the cell, the metal bindingprotein has specificity for a metal, and the first oligomer of the metalbinding protein expressed on the surface of the cell is capable ofaggregating with a second oligomer of the metal binding protein in thewater upon binding a metal.

In certain embodiments, the cell can be yeast.

In certain embodiments, the linker can be a monomer of the metal bindingprotein.

In certain embodiments, the second oligomer of the metal binding proteincan be secreted from the cell.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the metal can be nickel, iron, copper, cobalt,lead, cadmium or mercury.

In certain embodiments, the metal binding protein can be glutaminesynthetase.

In certain embodiments, the method can further include appending a highaffinity protein including to the metal binding protein, where the highaffinity protein including a metal binding domain, wherein the metalbinding domain has specificity for the metal.

In certain embodiments, the high affinity protein can be a plantmetallothionein.

In certain embodiments, the method can further include expressing themetal binding protein in a yeast host strain.

In another aspect, a method of mining a metal can include preparing acomposition including a cell and a first oligomer of a metal bindingprotein expressed on a surface of the cell via a linker, where thelinker can be tethered to the first oligomer of the metal bindingprotein and to a surface of the cell, the metal binding protein hasspecificity for a metal and the first oligomer of the metal bindingprotein expressed on the surface of the cell is capable of aggregatingwith a second oligomer of the metal binding protein in the water uponbinding a metal, contacting water with the composition and lysing thecell to obtain the metal.

In certain embodiments, the cell can be yeast.

In certain embodiments, the linker can be a monomer of the metal bindingprotein.

In certain embodiments, the second oligomer of the metal binding proteincan be secreted from the cell.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the transition metal can be nickel, iron,copper, cobalt, lead, cadmium or mercury.

In certain embodiments, the metal can be a noble metal.

In certain embodiments, the noble metal can be gold, silver or platinum.

In certain embodiments, the metal can be a rare-earth metal.

In certain embodiments, the rare-earth metal can be cerium (Ce),dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium(Ho), lanthanum (La), lutetium (Lu), neodymium (Nd), praseodymium (Pr),promethium (Pm), samarium (Sm), scandium (Sc), terbium (Tb), thulium(Tm), ytterbium (Yb) or yttrium (Y).

In certain embodiments, the metal binding protein can be glutaminesynthetase.

In certain embodiments, the metal binding protein can be fused with ahigh affinity protein including a metal binding domain, wherein themetal binding domain has specificity for the metal.

In certain embodiments, the high affinity protein can be a plantmetallothionein.

In certain embodiments, the metal binding protein can be expressed in ayeast host strain.

In another aspect, a composition for remediating a metal to treat watercan include a cell expressing a membrane metal transporter, wherein themembrane metal transporter has specificity for a metal, a vacuoletransporter and a metal sequestration protein.

In certain embodiments, the cell can be yeast.

In certain embodiments, an ubiquitination ligase can be deleted in theyeast.

In certain embodiments, the ubiquitination ligase can be BSD2.

In certain embodiments, the membrane transporter can be SMF1.

In certain embodiments, the vacuole transporter can be CCC1.

In certain embodiments, the metal sequestration protein can be aphytochelatin synthase.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the metal can be cadmium.

In certain embodiments, SMF1 can be mutated to be sensitive to themetal.

In certain embodiments, the metal can be strontium, lead or mercury.

In certain embodiments, SMF1 can be mutated to destroy primaryubiquitination sites.

In certain embodiments, the membrane transporter can be Sul1 or Sul2.

In certain embodiments, the metal can be chromate.

In certain embodiments, the membrane transporter can be CTR1.

In certain embodiments, the metal can be copper.

In certain embodiments, the membrane transporter can be ZRT1.

In certain embodiments, the metal can be zinc.

In certain embodiments, the membrane transporter can be FRE1.

In certain embodiments, the metal can be iron.

In another aspect, a method of remediating a metal to treat water caninclude preparing a composition include a cell expressing a membranemetal transporter, where the membrane metal transporter has specificityfor a metal, a vacuole transporter, and a metal sequestration protein,and contacting water with the composition.

In certain embodiments, the cell can be yeast.

In certain embodiments, an ubiquitination ligase can be deleted in theyeast.

In certain embodiments, the ubiquitination ligase can be BSD2.

In certain embodiments, the membrane transporter can be SMF1.

In certain embodiments, the vacuole transporter can be CCC1.

In certain embodiments, the metal sequestration protein can be aphytochelatin synthase.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the metal can be cadmium.

In certain embodiments, SMF1 can be mutated to be sensitive to themetal.

In certain embodiments, the metal can be strontium, lead or mercury.

In certain embodiments, SMF1 can be mutated to destroy primaryubiquitination sites.

In certain embodiments, the membrane transporter can be Sul1 or Sul2.

In certain embodiments, the metal can be chromate.

In certain embodiments, the membrane transporter can be CTR1.

In certain embodiments, the metal can be copper.

In certain embodiments, the membrane transporter can be ZRT1.

In certain embodiments, the metal can be zinc.

In certain embodiments, the membrane transporter can be FRE1.

In certain embodiments, the metal can be iron.

In another aspect, a method of mining a metal can include preparing acomposition including a cell expressing a membrane metal transporter,where the membrane metal transporter has specificity for a metal, avacuole transporter, and a metal sequestration protein, contacting waterwith the composition, and lysing the cell to obtain the metal.

In certain embodiments, the cell can be yeast.

In certain embodiments, an ubiquitination ligase can be deleted in theyeast.

In certain embodiments, the ubiquitination ligase can be BSD2.

In certain embodiments, the membrane transporter is SMF1.

In certain embodiments, the vacuole transporter can be CCC1.

In certain embodiments, the metal sequestration protein can be aphytochelatin synthase.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the transition metal can be nickel, iron,copper, cobalt, lead, cadmium or mercury.

In certain embodiments, the metal can be lithium.

In certain embodiments, the metal can be a noble metal.

In certain embodiments, the noble metal can be gold, silver or platinum.

In certain embodiments, the metal can be a rare-earth metal.

In certain embodiments, the rare-earth metal can be cerium (Ce),dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium(Ho), lanthanum (La), lutetium (Lu), neodymium (Nd), praseodymium (Pr),promethium (Pm), samarium (Sm), scandium (Sc), terbium (Tb), thulium(Tm), ytterbium (Yb) or yttrium (Y).

In another aspect, a composition for remediating a metal to treat watercan include a cell including a knocked-out enzyme required insulfate-assimilation pathway, where the cell has specificity forreactions against a metal.

In certain embodiments, the enzyme can be HOM2, MET2, MET17 or CYS4.

In certain embodiments, in the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the metal can be mercury, zinc, copper, cadmiumor lead.

In certain embodiments, a surface of the cell can be modified to displaya biomineralization peptide.

In certain embodiments, a surface of the cell can be modified to displaya degenerate sequence that is biased towards cysteine, histidine,glutamic and aspartic acid residues.

In another aspect, a method of making a composition for remediating ametal can include deleting an enzyme required in sulfate-assimilationpathway in a cell, wherein the cell has specificity for reactionsagainst a metal and culturing the cell in a medium supplemented withcysteine and/or methionine.

In certain embodiments, the enzyme can be HOM2, MET2, MET17 or CYS4.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the metal can be mercury, zinc, copper, cadmiumor lead.

In certain embodiments, the method can further include modifying asurface of the cell to display a biomineralization peptide.

In certain embodiments, the method can further include modifying asurface of the cell to display a degenerate sequence that are biasedtowards cysteine, histidine, glutamic or aspartic acid residues.

In certain embodiments, the method can further include culturing thecell in the medium supplemented with sodium sulfide.

In certain embodiments, the method can further include culturing thecell in the medium buffered to maintain a pH of the media above 4.

In another aspect, a method of forming a metal nanoparticle can includepreparing a composition comprising a cell including a knocked-out enzymerequired in sulfate-assimilation pathway, wherein the cell hasspecificity for reactions against a metal, contacting the compositionwith water, and purifying the metal nanoparticle from the cell.

In certain embodiments, the purifying the metal nanoparticle from thecell can include enzymatically digesting the cell wall, pelleting thecellular debris, and collecting the supernatant.

In certain embodiments, the metal nanoparticle can be HgS, CdS, ZnS orPbS.

In certain embodiments, the cell can be yeast.

In certain embodiments, the method can further include culturing theyeast in a growth medium with cysteine and/or methionine.

In certain embodiments, the method can further include tuning a size ofthe metal nanoparticle by changing a content of cysteine and/ormethionine.

In certain embodiments, the method can further include tuning aproduction rate of the metal nanoparticle by changing a content ofcysteine and/or methionine.

In another aspect, a method of remediating a metal to treat water caninclude preparing a cell including a knocked-out enzyme required insulfate-assimilation pathway, where the cell has specificity reactionsagainst for a metal and contacting water with the composition.

In certain embodiments, the enzyme can be HOM2, MET2, MET17 or CYS4.

In certain embodiments, the metal can be copper, cadmium or lead

In certain embodiments, a surface of the cell can be modified to displaya biomineralization peptide.

In certain embodiments, a surface of the cell can be modified to displaya degenerate sequence that is biased towards cysteine, histidine,glutamic or aspartic acid residues.

In another aspect, a method of mining a metal can include preparing acell including a knocked-out enzyme required in sulfate-assimilationpathway, where the cell has specificity reactions against for a metaland contacting water with the composition.

In certain embodiments, the enzyme can be HOM2, MET2, MET17 or CYS4.

In certain embodiments, the metal can be a divalent metal.

In certain embodiments, the metal can be a transition metal.

In certain embodiments, the transition metal can be mercury, zinc,copper, cobalt, cadmium or lead.

In certain embodiments, the metal can be a noble metal.

In certain embodiments, the noble metal can be gold, silver or platinum.

In certain embodiments, the metal can be a rare-earth metal.

In certain embodiments, the rare-earth metal can be cerium (Ce),dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium(Ho), lanthanum (La), lutetium (Lu), neodymium (Nd), praseodymium (Pr),promethium (Pm), samarium (Sm), scandium (Sc), terbium (Tb), thulium(Tm), ytterbium (Yb) or yttrium (Y).

In certain embodiments, a surface of the cell can be modified to displaya biomineralization peptide.

In certain embodiments, a surface of the cell can be modified to displaya degenerate sequence that is biased towards cysteine, histidine,glutamic or aspartic acid residues.

Other aspects, embodiments, and features will be apparent from thefollowing description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows analogies between physicochemical and engineered biologicalprocesses.

FIG. 2 shows yeast displayed+multiplier protein system.

FIGS. 3A-3C show structure of multiplier proteins (GS). FIG. 3A showscrystal structure and TEM images of GS. FIG. 3B shows controlled proteinaggregation of multiplier proteins (GS monomers). FIG. 3C showscontrolled protein aggregation of multiplier proteins (GS oligomers).

FIG. 4 shows SEM image of yeast with aggregated GS.

FIG. 5 shows metal uptake with yeast+GS.

FIG. 6 shows quantifying GS expression level using FACS.

FIG. 7 shows staining of membrane transporter SMF1, and vacuoletransporter CCC1.

FIG. 8 shows effects on engineering yeast transporters on cadmiumuptake.

FIG. 9 shows metal transport screening pipeline.

FIG. 10A shows a diagram illustrating plant phytochelatin TaPCS1 toenhance metal tolerance in yeast. FIG. 10B shows TaPCS1 enhances heavymetal tolerance.

FIG. 11 shows growth curve of engineered strains in 100 μM cadmium.

FIGS. 12A-12B show using sulfate permeases to uptake structurallysimilar chromate ions. FIG. 12A shows basic electron density mapping ofsulfate and chromate show a high degree of structural resemblance. FIG.12B shows overexpressing sulfate permeases Sul1 and 2 lead to increasechromate uptake.

FIG. 13A shows simplified diagram of yeast's sulfur pathway. FIG. 13Bshows crystalline metal sulfide particles (e.g. PbS) using ΔCYS4 strain.FIG. 13C shows crystalline metal sulfide particles (e.g. PbS) usingΔHOM2. FIG. 13D shows crystalline metal sulfide particles (e.g. PbS)using ΔMET17. FIG. 13E shows a deeper look of the particles. FIG. 13Fshows EDX—ratiometric analysis of the metal sulfide particles. FIG. 13Gshows XRD—material crystallinity of the metal sulfide particles.

FIG. 14 shows lead acetate strips (left) turn black in the presence ofgaseous sulfur.

FIGS. 15A-15C show the effects of media condition on sulfide production.FIG. 15A shows the absence or presence of cysteine or methionine eitherincreases (ΔCYS4) or decreases sulfide production (ΔHOM2, MET2, MET17).FIG. 15B shows the absence of cysteine both reduces the onset of sulfideproduction and increases sulfide production yield for ΔMET17. FIG. 15Cshows effects on nutrient conditions on sulfide production.

FIGS. 16A-16C show precipitation of metal sulfides usingsulfur-producing yeast. FIG. 16A shows cultures grown overnight with Cu,Cd, Zn, Pb, Hg produce metal sulfides which give observable colorchanges indicative of CuS, CdS, ZnS, PbS and HgS precipitation. FIG. 16Bshows analysis under TEM shows that metal sulfides such as CdSprecipitate on the cell wall. FIG. 16C shows photos of Cds and PbSnanoparticle synthesis with/without yeast.

FIG. 17 shows hypothesized schematic of peptide mediated metal sulfidemineralization.

FIG. 18 shows proof of concept screening of cadmium and copper metalsulfide formation using yeast display peptide libraries (left image).The amount of precipitated CdS can be modulated by displaying differentpeptides on the yeast surface. (right image) Likewise, colonies can bediscriminated by colony color density.

FIG. 19 shows solubility curves of metals at various pH values.

FIGS. 20A-20C shows examples of combining strategies 1-3. FIG. 20A showsbiomineralization can be initiated with released sulfur compounds thatsubsequently nucleate on displayed biomineralization peptides. FIG. 20Bshows high valent metals are reduced to divalent forms which arerecognized by divalent metal transporters such as ZIPs, CTRs, FTRs, etc.FIG. 20C shows transported metals compartmentalized by a variety ofmechanisms such as phytochelation, mineralization, or vacuolecompartmentalization.

FIG. 21 shows sequence map of DNA cassette containing AGA1, AGA2,protein of interest (POI), and TRP1marker. Annotations specify relevantsequences such as promoters, terminators, tags, etc. FIG. 21 disclosesSEQ ID NO: 9.

FIG. 22A shows sequences encoding MTs (or other proteins) can berationally designed or mutated to generate a library of mutants to betested for improved metal uptake, waste removal, or waste conversion.FIG. 22B shows libraries screened via yeast display for metal uptake, oralternatively tested against waste removal such as TCE. FIG. 22C showsselection based on optical, colorimetric, or chemical assays to screenfor the most efficacious binders or catalyst via high-throughputplatforms such as flow cytometry or automated plate readers.

FIG. 23A shows sequence map of GAL1 inducible metal transportersfollowed by a V5 epitope tang and a CYC1 transcription terminator. FIG.23B shows illustration representing metal uptake via yeast metaltransporters.

FIG. 24A shows that genes of yeast metal transporters for zinc (ZRTs),copper (CTRs), iron (FRE), etc. were over-expressed and expressions werequalitatively assessed using immunofluorescence by staining the V5epitope tag. FIG. 24B shows uptake studies of over-expressed metaltransporters for Cu(II) (blue), Zn(II) (red) and Mn(II) (purple).Highlighted bars indicate significant increase in uptake compared to WT(dashed line).

FIG. 25 shows flow diagram of using and recycling DIY yeast remediationpackets for heavy metal removal.

FIG. 26 shows a simple genetic switch turns on the production of thegreen fluorescent protein (GFP) to indicate the presence of metalcontaminants still present in the filtered drinking water.

FIGS. 27A-27C show yeast display of plant metallothioneins (MTs) todemonstrate copper uptake and enhanced survivability in coppersolutions. FIG. 27A shows four families of plant MT proteins were takenfrom Arabidopsis Thaliana and expressed in the AGA1 and AGA2 yeastdisplay system. All families of MTs showed increased uptake of copper(FIG. 27B) and increased survivability in copper solutions (FIG. 27C).

FIGS. 28A-28B show using Percoll density gradients to fractionate cloneswith improved metal uptake efficiency (see also FIG. 9). FIG. 28A showsrepresentative image of the mutant strain cell band that is higher indensity (lower in the gradient) than compared to wild-type. FIG. 28Bshows elemental analysis on collected cells from FIG. 28A showing arelative increase in metal uptake.

FIGS. 29A-29D show engineering metal transporters to enhance heavy metaluptake. FIG. 29A shows schematic of heavy metal uptake pathways inyeast. FIG. 29B shows micrographs demonstrating that metal transporterSMF1 can be engineered to improve cadmium uptake. FIG. 29C shows cadmiumuptake of WT yeast and engineered yeast strains in μM (top) and mgCd²⁺/g dry weight (DW, bottom). FIG. 29D shows SMF1 mutagenesis results.

FIG. 30 shows wine yeast engineered to limit hydrogen sulfideproduction.

FIGS. 31A-31D show engineering yeast strains and detecting hydrogensulfide production. FIG. 31A shows lead acetate strips giving a binarymeasurement on whether strains are capable of overproducing sulfur. FIG.31B shows hydrogen sulfide columns marked with scales can determinehydrogen sulfide produced in cultures. FIGS. 31C and 31D show 1 ppmresolution columns (yellow) undergo a purple change in the presence ofhydrogen sulfide, whereas 5 ppm resolution columns (white) undergo abrown change.

FIGS. 32A-32B show that yeast can generate sulfur to precipitate metalsout of solution. FIG. 32A cadmium removal using yeast sulfur productioneliminates 90±5% of cadmium in solution. FIG. 32B Precipitated CdSparticles excite at 350 nm and emit at 415 nm which is characteristic ofCdS quantum dot behavior.

FIGS. 33A-33B show cross section TEM images of yeast cells with CdSembedded in the cell wall. FIG. 33A shows images showing speckles ofcadmium sulfide on the yeast cell wall. FIG. 33B shows particles areapproximately 10-20 nm in diameter, with some encased in a biologicalshell which might be a consequence of cell wall encasing or during cellwall extraction.

FIGS. 34A-34B show established infrastructure to producewater-remediating yeast cultures for global applications. FIG. 34A showsthe engineered yeast can be stored as freeze-dried or compressedpackages, as they normally are in for consumer purposes, for long-termstorage and portability. FIG. 34B shows the beer and wine industry havealready developed a cheap and scalable source of yeast to scale theproduction of engineered strains for water remediation.

DETAILED DESCRIPTION

Many of the current industrial methods used for water treatment lackmetal specificity and produce significant amounts of secondary wastewhich has made waste treatment an unsustainable process. See GökhanEkrem Ustün, Seval Kutlu Akal Solmaz, and Aşkin Birgül. Regeneration ofindustrial district wastewater using a combination of Fenton process andion exchange—A case study. Resources, Conservation and Recycling,52(2):425-440, 2007, and MA Barakat. New trends in removing heavy metalsfrom industrial wastewater. Arabian Journal of Chemistry, 4(4):361-377,2011, each of which is incorporated by reference in its entirety. Thecommonly used methods, ion-exchange, absorption, and chemicalprecipitation, also known as physicochemical methods, also have a highcost barrier preventing easy adoption in developing areas which are morelikely to require intensive heavy metal treatment. See R K Rattan, S PDatta, P K Chhonkar, K Suribabu, and A K Singh. Long-term impact ofirrigation with sewage effluents on heavy metal content in soils, cropsand groundwater case study. Agriculture, Ecosystems & Environment,109(3):310-322, 2005, which is incorporated by reference in itsentirety.

Therefore, there needs to be more sustainable technologies for watertreatment that require methods beyond just physical and chemicaltechniques. With current bioengineering techniques this balance betweenconsumption and waste of metals can be exploited to favor metalaccumulation and conversion without causing toxic effects. See RobertWysocki and Markus J Tamás. How Saccharomyces cerevisiae copes withtoxic metals and metalloids. FEMS microbiology reviews, 34(6):925-951,2010, which is incorporated by reference in its entirety. Simpleorganisms such as yeast can be genetically modified to act as livingagents that sequester and remove waste from the environment. The addedbenefit is that yeast can be easily modified and self-propagate withminimal user intervention, making them a desirable engineerable platformwhich is cheap, scalable, and easily handled.

Current approaches to clean contaminated waters and landmasses are tosynthesize chelating or reactive molecules to sequester heavy metals andtoxic compounds. See Deshpande, Kiranmayi, et al., “Efficientsequestration and reduction of of hexavalent chromium with organosilicasol-gels,” Journal of Materials Chemistry 15.29 (2005): 2997-3004, andTang, Hao, et al., “Reductive dechlorination of activatedcarbon-adsorbed trichloroethylene by zero-valent iron: carbon aselectron shuttle,” Journal of environmental quality 40.6 (2011):1878-1885, each of which is incorporated by reference in its entirety.However, these methods are costly and complex in design and arethemselves prone to forming waste by-products. An alternative method canavoid these problems by using biological peptide chemistry and proteinengineering a facile, modular, and scalable platform that uses proteinbinding domains as waste-containment agents. An advantage overchemically synthesizing polymers or fabricating devices is thesimplicity of designing novel proteins using current gene editingtechnology, modularity (whereas chemicals and devices have to redo thecycle of theorization, construction and validation), and cost-efficientscalability when considering the large volumes of contaminated waters totreat. See Needels, Michael C., et al., “Generation and screening of anoligonucleotide-encoded synthetic peptide library,” Proceedings of theNational Academy of Sciences 90.22 (1993): 10700-10704, and Houghten,Richard A., et al., “Generation and use of synthetic peptidecombinatorial libraries for basic research and drug discovery,” (1991):84-86, each of which is incorporated by reference in its entirety.

On the other end of the spectrum, current research is pursuingbioremediation techniques that focus on using natural organisms toremove or consume waste, thereby providing an environmentally friendlierand natural alternative for managing waste, Historically, bioremediationwas first invented by George Robinson in the 1960s by firstdemonstrating the use of bacteria to degrade petroleum and otherhydrocarbon based pollutants. See Golding, Lynnea, “Bioremediation ofPesticides,” which is incorporated by reference in its entirety.However, the process in which organisms break down hazardous substancesinto less toxic or usable forms has been a universal concept in allliving organisms. Especially so, the management of metal concentrationand localization in cells plays an incredibly important role in cellularhomeostasis. Of all the species that have adapted to handle highlypolluted environments, the most highly studied are plants, fungi, andbacteria. See Juwarkar, Asha A., Sanjeev K. Singh, and Ackmez Mudhoo, “Acomprehensive overview of elements in bioremediation,” Reviews inEnvironmental Science and Bio/Teclmology 9.3 (2010): 215-288, and Ron,Eliora Z. and Eugene Rosenberg, “Biosurfactants and oil bioremediation,”Current opinion in biotechnology 13.3 (2002): 249-252, each of which isincorporated by reference in its entirety. However, given the diversityof plants and bacteria and their respective genes and proteins that playa role in heavy metal uptake bring into question whether using nativeorganisms with specific environmental and growth conditions is aforeseeable platform for a creating a scalable and accessiblebioremedation technology. Therefore, the next step should be in favor ofusing an engineerable host which can be manipulated to functionanalogously to plants and/or bacteria by expressing the relevantproteins using current technologies in molecular and geneticengineering.

Therefore, protein engineering can be applied on yeast to create thenext biological platform for bioremediation. Yeast have been a modelorganism for genetic studies since the age of Louis Pasteur, and sincethen yeast are becoming increasingly relevant in gene expression andprotein studies as genetic engineering technology continues to develop.The field of yeast biology has already optimized genetic manipulationtechniques such as transformations, genomic recombination, heterologousprotein expression, and design and function of genetic circuits. Inaddition, many of the studies that have identified phytochelatins,metallothioneins, metal transporters, and cytochromes were eitherdiscovered in yeast: or functionally identified using functionalcomplementation of yeast mutants proving that yeast already contain thebasic machinery for metal transport, uptake, and sequestration.Expressing the relevant proteins and enzymes can enable uptake andsequester metals beyond the normal limitations of wild-type yeast andvastly beyond the hazardous standards set by the EPA.

Disclosed herein is a method for remediating a metal to treat water orfor mining a metal to take the principles from ion-exchange, adsorption,and chemical precipitation and create analogous strategies in yeast. Theact of binding metals (ion-exchange), internalization (adsorption), andconversion (chemical precipitation) are naturally found in biologicalprocesses for cellular homeostasis. See Julian C Rutherford and Amanda JBird. Metal-responsive transcription factors that regulate iron, zinc,and copper homeostasis in eukaryotic cells. Eukaryotic cell, 3(1):1-13,2004, which is incorporated by reference in its entirety. In certainembodiments, genetically engineered yeast can be used as a means tobioremediate waste waters. In certain embodiments, a combination ofyeast display to bind metals onto the surface of yeast, over-expressionof metal transporters to uptake metals intracellularly, and biochemicalpathways that enables to recycle captured metals using metabolic enzymescan be used. In parallel, yeast enzymes can be developed to consumeand/or degrade harmful organic compounds such as TCE generated frommining and other environmentally unfriendly practices. Given currentbioengineering techniques, these mechanisms can be perturbed to favormetal accumulation by exposing more metal binding proteins, increasingactivity of metal transporters, or promoting specific metal reactionpathways, for example. This leads to the creation of three uniquestrategies: cell surface display, cell uptake, and biomineralization(FIG. 1): 1) cell surface display of metal binding proteins mimics themechanism of action of surface functionalized ion-exchange resins; 2)physical uptake of metals can be achieved by engineering hyperactivemetal transporters; and 3) yeast can supply reactive by-products tomineralize metals from solution, instead of relying on externalchemicals for chemical precipitation. Despite differing approaches, allthree can be optimized based on metal capture capacity, metalspecificity and selectivity, and yeast metal tolerance andsurvivability. In certain embodiments, the metal can be a divalentmetal. In certain other embodiments, the metal can be a transitionmetal.

These strategies closely mimic the mechanism of action of thephysicochemical methods mentioned above, yet address the limitations ofcost, development time, and scalability. It is possible to optimizethese strategies such that organisms, like yeast, can be used to treatand manage waste for the environment and the public.

Yeast Display Capture Capacity

Yeast display as a method for material capture is limited by celldensity and the expression number of peptide/proteins per cell surface.Nominal capture capacity is in the micromolar range assuming culturedensities from ten to one hundred thousand cells per milliliter,expression levels ranging from ten to one hundred thousand(experimentally determined), and effective binding sites perpeptide/protein between 1 and 10. Uptake can then be defined as:uptake [M]=N _(c) ×N _(e) ×n÷N _(A)Where

-   -   N_(c)=density of cells    -   N_(e)=expression number per cell    -   n=binding sites per expression    -   N_(A)=Avogadro's number (6.022e23)        Density Changes Due to Metal Uptake

Assuming transporter metal uptake of 100 μM (denoted as M; empiricallydetermined), and nominal values for yeast volume and density, thendensity changes due to metal uptake can be determined with:

${\Delta\rho} = {\rho + \frac{m_{metals}}{V}}$

Assuming volume does not increase dramatically with metalinternalization. The added mass contributed by the metals can becalculated withm _(metals) =M×V×MW÷N _(A)Where

-   -   ρ=yeast density (1.129 g/mL)    -   V=yeast volume (35×10⁻¹⁵ mL)    -   m_(metals)=added mass due to metal    -   MW=metal molecular weight    -   N_(A)=Avogadro's number (6.022e23)

Using a lower end molecular weight (MW) of 54.9 (manganese) and a higherend molecular weight of 207.2 (lead) density changes can range from2-25%.

Strategy 1—Cell Surface Capture—Increasing Capture Capacity of YeastDisplay Using “Multiplier” Proteins.

Strategy 1 is focused on overcoming the low metal capture capacity ofyeast display when compared to physicochemical techniques such asion-exchange.

A composition and a method for remediating a metal to treat water or formining a metal can include a cell and a first oligomer of a metalbinding protein expressed on a surface of the cell via a linker, wherethe linker is tethered to the first oligomer of the metal bindingprotein and to a surface of the cell, the metal binding protein hasspecificity for a metal; and the first oligomer of the metal bindingprotein expressed on the surface of the cell is capable of aggregatingwith a second oligomer of the metal binding protein in the water uponbinding a metal. The oligomer can be an oligonucleotide having 1 to 30nucleotides, for example, 3 to 25 nucleotides.

In certain embodiments, a “multiplier” protein system can be used, whereprotein monomers tethered to a metal binding protein (MBP) aggregateonto the yeast surface, effectively multiplying the number of MBPsdisplayed per cell. These yeast aggregates are able to capture 1-10 mMof copper, cobalt, and cadmium at 1 OD₆₀₀ of cells; 2-orders ofmagnitude greater than any existing techniques on yeast display captureof heavy metals. In certain embodiments, metal specificity can becontrolled by engineering the tethered MBPs. In certain embodiments,engineered plant metallothioneins can be used. Engineered plantmetallothioneins are small proteins with metal specificity towardsmercury, cadmium, lead, and a range of other divalent metals. In certainembodiments, the multiplier protein can be engineered to aggregate inresponse to various stimuli, or to be reversible, such that thisstrategy can both capture, and then release the collected metalcontaminates.

In certain embodiments, the metal can be a divalent metal. In certainother embodiments, the metal can be a transition metal.

Strategy 2—Metal Uptake—Creating Yeast Hyper Accumulators Using Membraneand Vacuole Transporters.

Strategy 2 exploits the yeast metal transport system to hyper accumulatemetals present in the environment.

A composition and a method for remediating a metal to treat water or formining a metal can include a cell expressing a membrane metaltransporter, where the membrane metal transporter has specificity for ametal, a vacuole transporter, and a metal sequestration protein. Thecomposition can be a component of a water treatment kit.

In certain embodiments, the membrane transporter is expressed on themitochondrial membrane. In certain embodiments, the membrane transporteris SMF1. In certain embodiments, the vacuole transporter is CCC1. Incertain embodiments, both membrane transporter, SMF1, and vacuoletransporter CCC1, can be used in combination to uptake heavy metals suchas cadmium. Compared to wild-type, cadmium uptake increased by 10-fold.In certain embodiments, SMF1 can be engineered to become sensitive toother metals such as strontium, lead, and mercury. In certainembodiments, conserved transmembrane domains can be identified throughglobal multi-alignments and mutagenized these portions to create SMF1libraries. These libraries can be tested against different metals andassayed based on density changes because of the mass increase due tometal accumulation.

In addition, to counter the toxicity effects of metal uptake, plantphytochelatin synthase, TaPCS1 can be expressed to increase metaltolerance. The combined expression of CCC1 and TaPCS1 allows yeast tosurvive at 100 μM cadmium, whereas wild-type yeast dies atconcentrations beyond 5 μM.

In certain embodiments, the metal can be a divalent metal. In certainother embodiments, the metal can be a transition metal.

Strategy 3—Metal Conversion—Using Yeast's Sulfur by-Products toPrecipitate Heavy Metals

Strategy 3 uses sulfur released from engineered yeast to react withmetals in solution.

A composition and a method for remediating a metal to treat water or formining a metal can include a cell including a knocked-out enzymerequired in sulfate-assimilation pathway, where the cell has specificityfor a metal.

In certain embodiments, enzymes required in the sulfate-assimilationpathway can be knocked out to retard the conversion of sulfates to thiolmetabolites allowing a build-up of hydrogen sulfide precursors. Inaddition, nutrient sources such as cysteine and methionine can be variedto affect the production rate and quantity of produced hydrogen sulfide.

Preliminary studies show sulfur accumulating up to 1 mM in culture whichreadily reacts with copper, cadmium, and lead. Investigation under TEMshows that reacted metal sulfides form consistently sized nanoparticlesin the range of 20-50 nm on the yeast cell wall. Particles are easilypurified by enzymatically digesting the cell wall, pelleting thecellular debri, and collecting the supernatant. Specifically, purifiedCdS particles exhibit a unique excitation and emission wavelength at 395and 430 respectively, characteristic of quantum dots in that size-range.The purity and quality of yeast generated quantum dots can be confirmedusing X-ray diffraction and TEM. In parallel, various conditions such aspH, media composition, and strain-type can be tested to understand theeffect on metal sulfide formation with respects to size, crystallinity,quantity, and monodispersity.

In certain embodiments, the metal can be a divalent metal. In certainother embodiments, the metal can be a transition metal.

1. Strategy 1—Cell Surface Capture

A paper by Ruta et. al. functionalized the yeast surface withhexapeptides to capture a range of common divalent metals such asnickel, copper, iron, etc. In addition, cells with displaying metalbinding proteins tend to be more metal tolerant, as metals capturedextracellularly are prevented from entering the cell body. See RobertWysocki and Markus J Tamás. How Saccharomyces cerevisiae copes withtoxic metals and metalloids. FEMS microbiology reviews, 34(6):925-951,2010, Lavinia Liliana Ruta, Ralph Kissen, Ioana Nicolau, Aurora DanielaNeagoe, Andrei Josá Petrescu, Atle M Bones, and Ileana CorneliaFarcasanu. Heavy metal accumulation by Saccharomyces cerevisiae cellsarmed with metal binding hexapeptides targeted to the inner face of theplasma membrane. Applied Microbiology and Biotechnology, pages 1-15,2017, and Oscar N Ruiz, Derry Alvarez, Gloriene Gonzalez-Ruiz, and CesarTorres. Characterization of mercury bioremediation by transgenicbacteria expressing metallothionein and polyphosphate kinase. BMCbiotechnology, 11(1):82, 2011, each of which is incorporated byreference in its entirety. However, current cell surface bindingcapacities, which are in the μM range, still cannot compete withion-exchange due to their extremely low capture capacity and poorcapture to cell weight ratio. See M A Barakat. New trends in removingheavy metals from industrial wastewater. Arabian Journal of Chemistry,4(4):361-377, 2011, and P Stathi, I T Papadas, A Tselepidou, and YDeligiannakis. Heavy-metal uptake by a high cation-exchange-capacitymontmorillonite: The role of permanent charge sites. Global nest,12(3):248-255, 2010, each of which is incorporated by reference in itsentirety. Therefore, the number of binding sites per cell is thegreatest limiting factor that limits the effectiveness of cell surfacedisplay.

1.1. Using Multiplier Proteins to Increase Metal Uptake Capacity

One method to address the limitation of cell surface display is todisplay multiple repeats of the same binding protein on a singledisplayed unit. Going one step further, one can instead aggregateproteins onto the yeast surface to create a metal capture network. Thismechanism of aggregating proteins onto the yeast surface can be achievedby a combination of displaying and secreting so called “multiplier”proteins, proteins that oligomerize to themselves. For example, anengineered yeast strain can display a single multiplier protein, and thesame, or another yeast strain, can secrete the same protein into themedia. In the presence of metal the secreted proteins can oligomerizeand inevitably anchor to the protein displayed on the yeast surface,thereby forming an aggregated network. This network anchored on theyeast surface effectively multiplies the expression level of thetypically single displayed protein on the yeast surface (FIG. 2). FIG. 2shows one cell displaying a protein oligomer, and another (or the samecell) can secrete protein monomers that aggregate onto the displayedprotein. Linkers fused with other proteins of interest (POI) can beappended to the oligomers to tailor the network's metal bindingproperties.

In certain embodiments, the multiplier protein used in this strategy canbe glutamine synthetase (GS for short; PDB: 2GLS), a bacterial proteinwhich has been studied for its role in glutamate and glutaminesynthesis. One unique property that has been somewhat overlooked is itsability to aggregate in a structurally unique pattern in the presence ofdivalent metals (FIGS. 3A-3C). In FIGS. 3A-3C, top images are renderedstructures of GS from crystollographic data. Bottom are TEM images of GSunits as well as aggregates. See Bennett M Shapiro and ER Stadtman.[130] glutamine synthetase (Escherichia coli). Methods in enzymology,17:910-922, 1970, which is incorporated by reference in its entirety.The rate of aggregation and the degree of oligomerization is dependenton exposure time and concentration of metal.

As a pilot-study, glutamine synthetase was overexpressed and purifiedfrom BL21 bacteria cells and added at 100 μM to the medium containingyeast displaying the same protein. Aggregation was visible by eye aswell as examined using scanning electron microscopy. The effect ofaggregation on metal uptake was quantitatively measured usinginductively coupled plasma (ICP). Species such as nickel, iron, andcopper had uptakes of 5-10 mM whereas cobalt, lead, and cadmium rangedfrom 1-5 mM given 1 OD600 of cells in synthetically defined media (CSM).The difference in binding capacity between species like iron and copperto the heavier and larger atoms such as lead and cadmium could be due todifferent GS binding affinities. In FIG. 5, top image shows (tubeslabeled left to right) solution of yeast, and yeast with 10 mM Cu, 10 mMCo, and 10 mM Cd. The right image shows the same conditions but with theaddition of 100 μM GS. Bottom figure shows metal uptake percentagequantified using ICP.

Without cells, glutamine synthetase alone is an effective metal binder.But with the addition of yeast, the aggregated network aggregates andsinks, simply due to the yeast's higher density allowing for easyseparation from treated waters. Another advantage is that the aggregatecan now be packed onto filtration or chromatography columns as membranefilters are typically sub-micron to micron which can easily excludeyeasts. So rather than handling liquids of culture and proteins, one caninstead package these aggregated complexes in filtration columns thatare easy to handle and use.

1.2. Tethering Metal Binding Proteins/Motifs for Increase MetalSpecificity

To further augment the binding capacity of this multiplier system,additional proteins containing metal binding domains can be fused ontoGS to increase binding capacity and tailor for metal specificity. Incertain embodiments, proteins such as plant metallothioneins can be usedbecause of their low molecular weight and high metal binding affinity aswell as their multiple binding domains (between 7-14). See ChristopherCobbett and Peter Goldsbrough. Phytochelatins and metallothioneins:roles in heavy metal detoxification and homeostasis. Annual review ofplant biology, 53(1):159-182, 2002, which is incorporated by referencein its entirety. Also, plant metallothioneins have a high affinity forother metals such as mercury and strontium that do not bind to GS. SeeGerald Henkel and Bernt Krebs. Metallothioneins: Zinc, cadmium, mercury,and copper thiolates and selenolates mimicking protein active sitefeatures-structural aspects and biological implications. Chemicalreviews, 104(2):801-824, 2004, and Ivo Fabrik, Jiri Kukacka, JiriBaloun, Ivo Sotornik, Vojtech Adam, Richard Prusa, David Vajtr, PetrBabula, and Rene Kizek. Electrochemical investigation ofstrontium-metallothionein interactions—analysis of serum and urine ofpatients with osteoporosis. Electroanalysis, 21(3-5):650-656, 2009, eachof which is incorporated by reference in its entirety.

In certain embodiments, these plant metallothioneins (MTs) can beappended to the N′ terminus of GS, as GS's main aggregating domain islocated at the C′ terminus (determined by analyzing the crystalstructure and using predicitve algorithms such as TANGO (see Ana-MariaFernandez-Escamilla, Frederic Rousseau, Joost Schymkowitz, and LuisSerrano. Prediction of sequence-dependent and mutational effects on theaggregation of peptides and proteins. Nature biotechnology, 22(10):1302,2004, which is incorporated by reference in its entirety). By tetheringplant MTs on the GS multiplier protein system, metal binding capacitiescan be at least doubled, and more so binding affinities can now favorheavier elements such as cadmium, lead, and mercury.

1.3. Additional Embodiments to Improve Secretion Yields

The biggest limitation of the current multiplier protein system is thelow expression levels of GS, both with respects to display andsecretion. Even with codon optimization, GS is displayed on less than20% of cells (FIG. 6), and secretion levels are barely detectable viaWestern Blot (not shown). FIG. 6 shows 2D histogram of FACS dataobserving fluorescently tagged N′-terminus HA tag (FITC,λex=488 nm) andC′-terminus Myc tag (CY5,λ_(ex)=647 nm).

Western blots of cell lysate show two bands with equal intensity, onewith the correct molecular weight of GS, and the larger being GS+signalpeptide. Therefore, there are two inefficiencies for GS export, thefirst being proper cleavage of the signal pep-tide, and the second isthe transport out of the cell after peptide cleavage.

In certain embodiments, S. cerevisiae's α-mating-factor can be used as asignal peptide.

In certain embodiments, other mating factors such as AGA1/2 and EXG1 canbe used to improve yield as they are processed through differentsecretion pathways possibly allowing easier passage and folding. SeeLars Ellgaard, Maurizio Molinari, and Ari Helenius. Setting thestandards: quality control in the secretory pathway. Science,286(5446):1882-1888, 1999, and Gunnar von Heijne. The signal peptide.Journal of Membrane Biology, 115(3):195-201, 1990, each of which isincorporated by reference in its entirety.

In certain embodiments, the expression process can be conducted at 20°C. (or lower) to improve proper GS folding in order to avoid the cell'sunfolded protein response which destroys poorly folded proteins in theendoplasmic reticulum, and is often a major problem for secretingheterologous proteins in yeast. See David Ron and Peter Walter. Signalintegration in the endoplasmic reticulum unfolded protein response.Nature reviews. Molecular cell biology, 8(7):519, 2007, and DagangHuang, Patrick R Gore, and Eric V Shusta. Increasing yeast secretion ofheterologous proteins by regulating expression rates and post-secretoryloss. Biotechnology and bioengineering, 101(6):1264-1275, 2008, each ofwhich is incorporated by reference in its entirety.

In other embodiments, GS can be expressed in Pichia Pastoris, a commonlyused yeast host strain for heterologous expression of prokaryotic andeukaryotic proteins that has a well defined secretory pathway.

In other embodiments, a different multiplier besides GS can be used. Theidentity of the protein is not a major concern just as long as it can besufficiently displayed and secreted. Another survey can be done throughthe literature and the protein data bank (www.rcsb.org/pdb/home/home.do,which is incorporated by reference in its entirety). To identify asuitable candidate, a multiplier protein must have well characterizedand controllable aggregating properties. These aggregating propertiesmust remain when fused to another protein (e.g. plant MTs) either at theN′ or C′ terminus and secrete more efficiently than GS.

2. Strategy 2—Metal Uptake

Plants have evolved a unique ability to tolerate heavily contaminatedsoils, specifically heavy metals such as cadmium, arsenic, chromium,etc. See Nicoletta Rascio and Flavia Navari-Izzo. Heavy metalhyperaccumulating plants: how and why do they do it? and what makes themso interesting? Plant science, 180(2):169-181, 2011, and MajetiNarasimha Vara Prasad and Helena Maria de Oliveira Freitas. Metalhyperaccumulation in plants: biodiversity prospecting forphytoremediation technology. Electronic journal of biotechnology,6(3):285-321, 2003, each of which is incorporated by reference in itsentirety. Researchers have attributed this unique ability to acombination of hyper-active metal transporters and a variety ofmetal-binding proteins that uptake and guard against metal poisoning.See Stephan Clemens, Michael G Palmgren, and Ute Krämer. A long wayahead: understanding and engineering plant metal accumulation. Trends inplant science, 7(7):309-315, 2002, which is incorporated by reference inits entirety. Strategy 2 utilizes a parallel mechanism to that of plantsby endowing yeast strains with hyperactive membrane and vacuoletransporters, as well as promiscuous metal binding proteins to createyeast hyperaccumulators that internalize large amounts of metals.

2.1. Expressing Relevant Transporters and Proteins to AchieveHyperaccumulating Activity

Much like plants, the requirements for metal hyperaccumulation in yeastare: 1) membrane metal transporters; 2) vacuole transporters; and 3)metal sequestration proteins to increase metal tolerance.

Out of 16 transporters screened, yeast SMF1 was chosen because of itswell-studied mechanism of action in addition to its ability to transporta variety of divalent metals. See P Courville, R Chaloupka, and MFMCellier. Recent progress in structure-function analyses of nrampproton-dependent metal-ion transporters this paper is one of a selectionof papers published in this special issue, entitled csbmcbmembraneproteins in health and disease. Biochemistry and Cell Biology,84(6):960-978, 2006, which is incorporated by reference in its entirety.most metal transporters are heavily regulated by proteases andubiquitinases to balance the concentration of intracellular metals. SeeElina Nikko, James A Sullivan, and Hugh R B Pelham. Arrestin-likeproteins mediate ubiquitination and endocytosis of the yeast metaltransporter smf1. EMBO reports, 9(12):1216-1221, 2008, and StevenLam-Yuk-Tseung, Gregory Govoni, John Forbes, and Philippe Gros. Irontransport by nramp2/dmt1: ph regulation of transport by 2 histidines intransmembrane domain 6. Blood, 101(9):3699-3707, 2003, each of which isincorporated by reference in its entirety. The major SMF1 ubiquitinationligase, BSD2, was deleted (see Xiu Fen Liu and Valeria Cizewski Culotta.Post-translation control of nramp metal transport in yeast role of metalions and the bsd2 gene. Journal of Biological Chemistry,274(8):4863-4868, 1999, which is incorporated by reference in itsentirety) with no signs of compromising yeast health. In addition,lysine residues K33,34 in SMF1 were mutated to arginines to destroy theprimary ubiquitination sites of SMF1 that are recognized by otherubiquitination ligases. See Elina Nikko, James A Sullivan, and Hugh R BPelham. Arrestin-like proteins mediate ubiquitination and endocytosis ofthe yeast metal transporter smf1. EMBO reports, 9(12):1216-1221, 2008,which is incorporated by reference in its entirety. The engineered SMF1is referred to as SMF1* (or SMF1-star).

Finally, a vacuole transporter was chosen by co-expressing selected oneswith SMF1* and choosing the vacuole transporter that enhanced the uptakeof cadmium. In FIG. 7, blue is DAPI nuclear stain. SMF1 appended with aV5 tag was stained with AlexaFluor 488 (green), and CCC1 was appendedwith a ag tag was stained with AlexFluor 647 (red). The best performingcandidate was CCC1, normally a Fe²⁺ and Mn²⁺ transporter, which showedmore than 3-fold increase in cadmium uptake (FIG. 8).

2.2. Mutating and Screening of SMF1 for Changes in Metal Specificity andSelectivity

One of the biggest limitations to rationally engineering metaltransporters is the lack of structural information due tocrystallization difficulty and the inability to reconstitutemembrane-like environments ex-vivo. See Elisabeth P Carpenter,Konstantinos Beis, Alexander D Cameron, and So Iwata. Overcoming thechallenges of membrane protein crystallography. Current opinion instructural biology, 18(5):581-586, 2008, which is incorporated byreference in its entirety. Therefore, most structure-to-functioninformation is obtained via meticulous and often times tedious pointmutations in hypothesized residues. Many of these studies haveidentified significant transmembrane domains, yet most of the resultslead to a loss of function. See P Courville, R Chaloupka, and MFMCellier. Recent progress in structure-function analyses of nrampproton-dependent metal-ion transporters this paper is one of a selectionof papers published in this special issue, entitled csbmcbmembraneproteins in health and disease. Biochemistry and Cell Biology,84(6):960-978, 2006, Steven Lam-Yuk-Tseung, Gregory Govoni, John Forbes,and Philippe Gros. Iron transport by nramp2/dmt1: ph regulation oftransport by 2 histidines in transmembrane domain 6. Blood,101(9):3699-3707, 2003, and J M Argüello. Identification ofion-selectivity determinants in heavy-metal transport p1b-type atpases.The Journal of membrane biology, 195(2):93-108, 2003, each of which isincorporated by reference in its entirety.

Fortunately, SMF1 has significant homology and phylogeneticrelationships with a large class of divalent transporters, namely Nramps(natural resistance-associated macrophage proteins) and DMTs (divalentmetal transporters). See Ute Krämer, Ina N Talke, and Marc Hanikenne.Transition metal transport. FEBS letters, 581(12):2263-2272, 2007, whichis incorporated by reference in its entirety. Because of this,researchers have discovered conserved regions that are necessary forSMF1 function. Through sequence analysis researchers have hypothesizedthat transmembrane domains 1-4, 5-6, and 9 are responsible fordiscriminating between and facilitating metal transport.

In order to further narrow the number of significant domains, a globalmulti-alignment analysis with 81 of the closest ranked members similarto SMF1 was performed. The 81 members were filtered from a potentiallist of >14,000 sequences queried based on name searches from Uniprot(www.uniprot.org/) by using Clustal Omega(www.ebi.ac.uk/Tools/msa/clustalo/). The 81 members were globallyaligned and spans of homology were quantified using Shannon entropy(Equation 1) which is a simple and direct metric for identifyingconserved protein regions. See William S J Valdar. Scoring residueconservation. Proteins: structure, function, and bioinformatics,48(2):227-241, 2002, which is incorporated by reference in its entirety.

$\begin{matrix}{{H(X)}_{j} = {\sum\limits_{i = 0}^{n}\;{{P\left( x_{i} \right)}\log_{2}{P\left( x_{i} \right)}}}} & (1)\end{matrix}$

Rows of aligned sequences (i) are scored per residue with respects tothe queried sequence (j) by calculating the probability (P) of thatresidue's appearance in the global alignment according to the Shannonentropy function. The lowest entropic score (most conserved) regionswere transmembrane domain 1 and 6. Both domains were amplified usingerror-prone PCR and homologously recombined into SMF1 to generate alibrary of mutants.

Developing High Throughput Screening of Mutated SMF1 Library

Mutants were screened by subjecting libraries to 100 μM metal ions(Me²⁺) in culture and fractionated based on density changes. Changes indensity were used to assess differences in metal uptake, as the uptakeof metals imparts additional mass to the cell. See William H Grover,Andrea K Bryan, Monica Diez-Silva, Subra Suresh, John M Higgins, andScott R Manalis. Measuring single-cell density. Proceedings of theNational Academy of Sciences, 108(27):10992-10996, 2011, which isincorporated by reference in its entirety. Cells were separated usingdensity gradient centrifugation; the furthest migrated layers in thedensity gradient were manually selected, re-plated, sequenced, andquantitatively tested for metal uptake using ICP. These rounds wererepeated 3 times for cadmium, and are ongoing for elements such asstrontium and lead.

In FIG. 9, SMF1 was mutagenized using error-prone PCR and assayed formetal uptake using density gradient centrifugation. Cells that uptakethe most metals migrate furthest to the bottom. Cells were isolated witha syringe, then plated, picked, sequenced, and finally confirmed formetal uptake using ICP.

2.3. Increasing Metal Tolerance & Survival

Along with hyperactive metal transporters, plants can tolerate unusuallyhigh metal concentrations by arming themselves with a variety of metalbinding proteins, namely glutathiones and metallothioneins. SeeChristopher Cobbett and Peter Goldsbrough. Phytochelatins andmetallothioneins: roles in heavy metal detoxification and homeostasis.Annual review of plant biology, 53(1):159-182, 2002, which isincorporated by reference in its entirety. Plants augment this layer ofdefense by oligomerizing glutathiones into phytochelatins which act as anetwork to internalize metals into compartmentalized areas. Id. Clemenset. al. found that a common wheat phytochelatin synthase, TaPCS1,dramatically enhanced metal tolerance when expressed in yeast. SeeStephan Clemens, Eugene J Kim, Dieter Neumann, and Julian I Schroeder.Tolerance to toxic metals by a gene family of phytochelatin synthasesfrom plants and yeast. The EMBO journal, 18(12):3325-3333, 1999, whichis incorporated by reference in its entirety. Performing a similarexperiment, a constitutive overexpression of TaPCS1 directly cloned fromwheat to wild-type yeast improved cadmium tolerance by almost 20-fold(FIGS. 10A-10B). FIG. 10A shows a diagram illustrating plantphytochelatin TaPCS1 to enhance metal tolerance I yeast. See Clemens,S., Kim, E. J., Neumann, D. & Schroeder, J. I. Tolerance to toxic metalsby a gene family of phytochelatin synthases from plants and yeast. TheEMBO Journal 18, 3325-3333 (1999), which is incorporated by reference inits entirety. In FIG. 10B, cultures were grown in increasing amounts ofcadmium levels (0-100 μM) and measured periodically at OD600. Growthrate k were extracted from curves using the logistic growth function.The left figure are wild-type yeast, the right figure are wild-typeyeast constitutively expressing wheat phytochelatin synthase TaPCS1.

2.4. Additional Embodiments

In certain embodiments, SMF1 transporters can be created to be selectivenot only to cadmium, but also for additional metals such as strontium,lead, mercury, etc. by focusing on filtering out libraries of SMF1mutants based on increase metal uptake, metal selectivity (KD) andspecificity between metals. For example, interference experiments candetermine whether mutants can uptake cadmium in the presence of excessmanganese (the preferred metal). Likewise, an experiment for metalselectivity can be executed to characterize titration curves in order todetermine the KD response of a given metal using colorimetric assays orICP.

Uptake of Negatively Charged Metal Compounds

Not all heavy metals are positively charged, there exist polyatomicmetal compounds in the negative state such as chromate (CrO₄ ²⁻) andarsenate (AsO₄ ²⁻) which are acutely toxic, however unrecognized bySMF1. See AD Dayan and AJ Paine. Mechanisms of chromium toxicity,carcinogenicity and allergenicity: review of the literature from 1985 to2000. Human & experimental toxicology, 20(9):439-451, 2001, and MichaelF Hughes. Arsenic toxicity and potential mechanisms of action.Toxicology letters, 133(1):1-16, 2002, each of which is incorporated byreference in its entirety. Yet, there exist permeases, much like metaltransporters, that facilitate the flux of basic nutrients such assulfates (SO₄ ²⁻) and phosphates (PO₄ ²⁻) into the cell. See BrunoAndré. An overview of membrane transport proteins in Saccharomycescerevisiae. Yeast, 11(16):1575-1611, 1995, which is incorporated byreference in its entirety. Given the structural similarity betweenchromates and sulfates, and between arsenates and phosphates (FIGS.12A-12B), it may be possible to “hijack” these permeases for chromateand arsenate uptake. As a preliminary experiment, both sulfate permeasesSul1, and Sul2 were tested for chromate uptake. As hypothesized,chromate uptake was elevated in yeast expressing Sul1, Sul2, but not tothe degree at which overexpressing wild-type SMF1 uptakes divalentmetals. One explanation is that chromate is much more acutely toxic thancadmium; at concentrations above 20 μM yeast die and no longer transportmetals. Another explanation is that permeases are selective enough todiscern between sulfates and chromates. Or perhaps the sulfateconcentration in yeast media overwhelms the transport of chromate.

Applications in Mining

Admittedly, cellular uptake of heavy metals has the least per celluptake capacity ratio than Strategy 1. However, a main advantage ofmetal transporters is that they are more sensitive to low amounts ofmetals, and are more metal specific. Besides SMF1, there exist otherselective metal transporters such as CTR1 (copper), ZRT1 (zinc), andFRE1 (iron). These transporters are able to recognize and uptake μM tonM amounts of metals despite the presence of other more concentratedions such as sodium and calcium naturally found in growth media. SeeStephan Clemens, Michael G Palmgren, and Ute Krämer. A long way ahead:understanding and engineering plant metal accumulation. Trends in plantscience, 7(7):309-315, 2002, which is incorporated by reference in itsentirety. This metal specific uptake can be capitalized to mine usefulmetals from waste water in addition to providing a mechanism for metalremoval. With the growing demand of electronically relevant metals suchas lithium, noble metals (gold, silver, platinum), and rare-earthmetals, mining operations are becoming exceedingly more dangerous andharmful to the environment. See Gavin Hilson. Pollution prevention andcleaner production in the mining industry: an analysis of currentissues. Journal of Cleaner Production, 8(2):119-126, 2000, which isincorporated by reference in its entirety. Likewise, metal extractiontypically takes several rounds of heating and smelting to purify asingle element, which is labor intensive and costly. See Jirang Cui andLifeng Zhang. Metallurgical recovery of metals from electronic waste: Areview. Journal of hazardous materials, 158(2):228-256, 2008, and A MAlfantazi and RR Moskalyk. Processing of indium: a review. MineralsEngineering, 16(8):687-694, 2003, each of which is incorporated byreference in its entirety. Given these limitations it may be possible toengineer yeast with specialized metal transporters to act as miningagents that harvest and concentrate scarce amounts of metals fromenvironmental sources under ambient conditions.

One example would be mining for lithium, noble metals (such as gold,silver, platinum) and rare-earth metals (such as cerium (Ce), dysprosium(Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho),lanthanum (La), lutetium (Lu), neodymium (Nd), praseodymium (Pr),promethium (Pm), samarium (Sm), scandium (Sc), terbium (Tb), thulium(Tm), ytterbium (Yb) or yttrium (Y)). A report by MIT projects thatlithium shortages will occur by 2050, yet some researchers acknowledgethat this can be avoided if the ocean were mined instead. See RichardMartin, As Demand for Lithium Grows, the Race to Extract It Intensifies,2015, which is incorporated by reference in its entirety. Despite anoverall massive quantity, approximately 1.5 trillion tons, lithiumconcentrations are low, ranging from 0.1-1 ppm (sub micromolar), anddifferentiating between the more abundant salt content makes lithiummining almost impossible. See Ernest E Angino and Gale K Billings.Lithium content of sea water by atomic absorption spectrometry.Geochimica et Cosmochimica Acta, 30(2):153-158, 1966, which isincorporated by reference in its entirety. A similar situation poses achallenge for rare-earth mining. Ironically, some rare-earths are moreabundant than more known elements such as cobalt and manganese, yet theyare typically difficult to extract because of the overwhelming presenceof iron, copper, and nickel compounds in mining ores. See Xiaoyue Du andThomas E Graedel. Global in-use stocks of the rare earth elements: afirst estimate. Environmental science & technology, 45(9):4096-4101,2011, which is incorporated by reference in its entirety. Again, highlyspecific yeast transporters could help differentiate rare-earths fromother elements and aid in the mining process. Yeast can effectively actas concentrators, specializing and storing desired metals of interest.Yeast can then be harvested and lysed to obtain the stored metal.Afterwards, simpler and more straightforward physicochemical techniquescan be used to isolate and purify these metals rather than having tosmelt and reheat ores common of traditional methods.

3. Strategy 3—Metal Conversion

The type of metal as well as its electronic state are equally importantin determining the metals' toxicity. For example, Cr(VI) is highlymutagenic and acutely toxic, whereas Cr(III) readily forms stable oxidesand precipitates out of solution. See Olga Muter, Aloizij s Patmalnieks,and Alexander Rapoport. Interrelations of the yeast Candida utilis andcr (vi): metal reduction and its distribution in the cell and medium.Process Biochemistry, 36(10):963-970, 2001, which is incorporated byreference in its entirety. Therefore, it is just as important to convertmetals to more benign electronic states as it is to capture and removethem from waste waters. However, what has limited bio-facilitatedconversion and reactions of heavy metals is the burden of supplyingelectron-rich molecules (e.g. pyruvates and NADP(H)s), which arethemselves rarely free in the cell other than for highly regulatedbiological processes. Even if these biomolecules are present, theelectrons must overcome a large activation barrier for convertingnormally stable metal ions to a more benign state.

3.1. Encouraging Sulfur Production in Yeast

Normally, cells are unable to process large amounts of metals from theenvironment; however, there exist a unique class of archaebacteria thatcan convert a select set of metals and organic compounds to otherelectronic states (e.g. Fe²⁺

Fe³⁺) for metabolic purposes. See Derek R Lovley. Dissimilatory metalreduction. Annual Reviews in Microbiology, 47(1):263-290, 1993, andKarrie A Weber, Laurie A Achenbach, and John D Coates. Microorganismspumping iron: anaerobic microbial iron oxidation and reduction. NatureReviews Microbiology, 4(10):752-764, 2006, each of which is incorporatedby reference in its entirety. For example, a class of bacteria, known assulfate-reducins, obtains energy by reducing sulfate (SO₄ ²⁻) tohydrogen sulfide (H₂S), and in the process gain energetic electrons forother cellular functions. Because of the production of hydrogen sulfide,these sulfate-reducins are able to erode iron and copper comprisedrocks, and have unfortunately become a hazard to old concrete and metalinfrastructures due to accelerated sulfur corrosion. See Washington AHamilton. Sulphate-reducing bacteria and anaerobic corrosion. AnnualReviews in Microbiology, 39(1):195-217, 1985, which is incorporated byreference in its entirety. However researchers have deliberately usedthis phenomenon to remove rusted metals from old mines and drains forcleaning purposes. See C Garcia, D A Moreno, A Ballester, M L Blazquez,and F Gonzalez. Bioremediation of an industrial acid mine water bymetal-tolerant sulphate-reducing bacteria. Minerals Engineering,14(9):997-1008, 2001, which is incorporated by reference in itsentirety. Given what is known about the scarcity of electron richspecies in the cell, sulfur lends itself extremely well as a metalreactant that is biologically generated. If production can becontrolled, sulfur could be utilized as a reliable source for heavymetal remediation.

Effects of Knockouts

Rather than using a difficult-to-culture sulfur-reducin, Strategy 3 isfocused on creating sulfur-producing yeast. Surprisingly, the wineindustry has studied the effects of yeast related-sulfur production withrespect to wine quality. For the past century, winemakers have realizedthat over-fermentation, or failing to supply sufficient nutrientsources, causes yeast to produce a pungent smell during wine-making. SeeCarla S Thomas, Roger B Boulton, Michael W Silacci, and W DouglasGubler. The effect of elemental sulfur, yeast strain, and fermentationmedium on hydrogen sulfide production during fermentation. Americanjournal of enology and viticulture, 44(2):211-216, 1993, which isincorporated by reference in its entirety. With the aid of recentmolecular biology techniques, researchers have discovered that due toextreme culture conditions essential proteins in the sulfate reducingpathway are either inhibited or denatured causing a buildup of sulfide(H²⁻) precursors. See J H Swiegers and I S Pretorius. Modulation ofvolatile sulfur compounds by wine yeast. Applied Microbiology andBiotechnology, 74(5):954-960, 2007, which is incorporated by referencein its entirety.

Enzymes in the sulfate assimilation pathway were knocked out to force abuildup of H₂S (FIGS. 13A-13G). In FIGS. 13A-13G, italicized enzymeswere knocked out and screened for sulfur production. Italicized andbolded enzymes are knockouts that produced a detectable amount ofsulfur. All others are necessary enzymes that are required for sulfurmetabolism. See Angela L Linderholm, Carrie L Findleton, GagandeepKumar, Yeun Hong, and Linda F Bisson. Identification of genes affectinghydrogen sulfide formation in Saccharomyces cerevisiae. Applied andenvironmental microbiology, 74(5):1418-1427, 2008, Chien Huang, MiguelRoncoroni, and Richard C Gardner. Met2 affects production of hydrogensulfide during wine fermentation. Applied microbiology andbiotechnology, 98(16):7125-7135, 2014, and Chien-Wei Huang, Michelle EWalker, Bruno Fedrizzi, Miguel Roncoroni, Richard C Gardner, andVladimir Jiranek. The yeast tum1 affects production of hydrogen sulfidefrom cysteine treatment during fermentation. FEMS yeast research, 16(8),2016, each of which is incorporated by reference in its entirety.Knockouts such as ΔMET2,17 and ΔHOM2 produced an observable amount ofsulfur determined by lead acetate strip and sulfur-displacement columns(FIG. 15A), whereas some deletions such as ΔCYS4 and ΔSER1,2 producedcysteine and methionine auxotrophy. Multi-deletions (2 & 3 knockouts)produced sick strains that barely formed colonies even on YPD (yeastpeptone dextrose media), and therefore were not tested.

Effects of Media Composition and Nutrients

The size and crystal properties of these nanoparticles can be tuned bychanging the nutritional content of the yeast culture, primarilycysteine and methionine precursors, thereby changing the production rateand timing of sulfur, which ultimately effects the kinetics of particlegrowth. More so, if these particles are properly made, they also havefluorescent properties (FIG. 32B), which makes production of thesecomplex particles autonomous, tunable, and cheap.

FIG. 14 shows a more quantitative detection method is to use columnsthat change color at a given height as a function of sulfur production.Rich culture sources such as YPD allowed all knockouts to grow, yet someproduced little-to-no sulfide. Compared to CSM media (completesupplement mixture, which lacks cysteine and contains minimalmethionine), ΔMET2,17 and ΔHOM2 produced sulfide at levels up to 100 ppm(FIG. 15A); however mutants such as ΔCYS4 and ΔSER1,2 failed to grow dueto cysteine or methionine auxotrophy.

Supplementing cysteine to CSM rescues ΔCYS4 allowing a production of 102ppm whereas ΔMET2,17 and ΔHOM2 produce almost half. Likewise, addingmore methionine to CSM reduces ΔMET2,17 and ΔHOM2 production by half. Anexplanation for the nutrient effects on sulfide production is that theaddition of cysteine or methionine eliminates the stress for thiolbiosynthesis which reduces sulfide production (FIG. 15C). Conversely,the addition of cysteine allows ΔCYS4 to grow, and since ΔCYS4 is thebiggest roadblock for cysteine synthesis (hence the auxotrophy), thesulfate pathway terminates at hydrogen sulfide gas which is therebyreleased.

3.2. Yeast Induced Metal Sulfide Precipitation

Metal sulfides have an extremely low solubility constant in solutions pHadjusted between 4-10. See DigitalAnalysisCo. Heavy Metal Reduction fromIndustrial Wastewater Streams, 2016, which is incorporated by referencein its entirety. Because of this, industrial chemical precipitationsometimes uses sulfur to treat highly contaminated waste water. However,the volatility and storage of sulfur becomes a hazard, so manyindustrial sites instead opt for sodium hydroxide. See Eddy Metcalf.Wastewater engineering: Treatment, disposal, reuse, metcalf & eddy.Inc., McGraw-Hill, New York, 2003, which is incorporated by reference inits entirety. Sulfide-producing yeast, however, is not burdened bypractical issues of sulfur management because the yeast itself can beeasily packaged and stored. Storage of sulfur simply requires thestorage of yeast that produces it. Furthermore, sulfide production canbe easily regulated depending on the demand by controlling nutrientconditions and gene expressions in the sulfate assimilation pathway.

Producing Cd, Cu Particles

ΔCYS4, ΔHOM2, and ΔMET2,17 mutants are able to precipitate approximately1 mM Cu²⁺ and 100 μM Cd²⁺ in CSM (ΔCYS4 cultures supplemented withcysteine).

Cross-sectional examination of metal precipitated cells usingcryo-sectioning and TEM show that CuS and CdS particles precipitate onthe cell wall. Simple lysis of the cell wall releases these particleswhich consistently range between 20-50 nm in diameter.

3.3. Using Yeast Display to Control Metal Sulfide Formation

Crystalline and structured metal sulfides are valuable for theirapplications in electronics, material fabrication, and optics. SeeJagadese J Vittal and Meng Tack Ng. Chemistry of metal thio- andseleno-carboxylates: precursors for metal sulfide/selenide materials,thin films, and nanocrystals. Accounts of chemical research,39(11):869-877, 2006, which is incorporated by reference in itsentirety. Compounds such as CdS, PbS, ZnS are routinely used for opticsand quantum dot synthesis. See A Mews, A Eychmüller, M Giersig, DSchooss, and H Weller. Preparation, characterization, and photophysicsof the quantum dot quantum well system cadmium sulfide/mercurysulfide/cadmium sulfide. The Journal of Physical Chemistry,98(3):934-941, 1994, which is incorporated by reference in its entirety.

Yeast are able to uniformly precipitate CuS, CdS, ZnS, PbS, and HgS;however, the mechanism is currently unknown, yet may be due tointeractions with the cell wall. In certain embodiments, particleformation can controlled via changing the surface chemistry of the cellwall by displaying peptides using yeast display. There are already knownpeptides that can mineralize the formation of CdS and ZnS, which hasbeen done on the M13 bacteriophage coat protein by the Belcher Lab. SeeChuanbin Mao, Christine E Flynn, Andrew Hayhurst, Rozamond Sweeney, JifaQi, George Georgiou, Brent Iverson, and Angela M Belcher. Viral assemblyof oriented quantum dot nanowires. Proceedings of the National Academyof Sciences, 100(12):6946-6951, 2003, which is incorporated by referencein its entirety.

Designing Metal Sulfide Biomineralization Libraries

A straightforward approach is to take known metal sulfide mineralizationpeptides and test for mineralization. However, the current literature islimited to only a few peptides that facilitate mineralization in extremebuffer conditions not amenable to yeast cultures (e.g. high pH, low saltcontent, and typically hazardous reducing agents). Moreover, thesepeptides solely focus on CdS, PbS, or CuS which can already be formedwith sulfur-producing yeast.

Therefore, new metal sulfide biomineralization peptides have beenself-generated by creating a yeast display library with degeneratesequence (NDN-NNK)_((8,12,16)) (SEQ ID NOS 1-3) (subscripts denotingrepeats) that are biased towards cysteine, histidines, glutamic andaspartic acid residues. These libraries are inserted into a yeastdisplay expression vector with the canonical yeast display AGA1 and AGA2cassette (plasmid named pYAGA). Such libraries can be generated andmutants can be visually screened based on metal sulfide color changeseither in cultures or on plates supplemented with the metal of interest(FIG. 18). More quantitative screening can be investigated using ICP andTEM.

3.4. Additional Embodiments

In certain embodiments, sulfide biomineralization yeast can beengineered and culture conditions can be optimized to control for metalsulfide particle formation.

In certain embodiments, yeast-synthesized metal sulfides can be used foreither materials or electronics.

Investigating pH Effects on Fe, Pb, Zn, Etc. Particle Formation

Adding 100 μM sodium sulfide (Na₂S) to yeast media readily precipitatescopper, cadmium, as well as iron, lead, mercury and zinc. However,metals other than copper or cadmium are very difficult to precipitatewith sulfur-producing yeast despite a similar buildup of producedsulfide. One hypothesis is that the rate of sulfur production is tooslow. Meaning, the rate of sulfide escaping from the media as a gas isfaster than the rate of reaction between metal and sulfide for the moresoluble metals such as iron and zinc. And as a function of time, after12-16 hours of yeast growth the culture pH can drop to 2, at which thepH is outside the permissible solubility range of most metal sulfideformation (FIG. 19). In FIG. 19, solubility/dissociation products andcalculations were taken from online resources. See Renata Bellová,Danica Melicherčiková, and Peter Tomčik. Calculation of conditionalequilibrium in serial multiple precipitation of metal sulfides withhydrogen sulfide stream generated from sodium sulfide: A didactic toolfor chemistry teaching. Quimica Nova, 39(6):765-769, 2016, which isincorporated by reference in its entirety. Therefore, a slow sulfideproduction rate can accumulate slower than the drop in pH and preventmetal sulfide formation. A possible solution is to simply bufferingyeast media to >pH 7 prior to inoculation to maintain the pH of themedia above 4.

In summary, three unique strategies can utilize yeast for heavy metalremediation. The first employs cell surface display in combination withprotein cell secretion to aggregate multiple repeats of the same proteinon the yeast surface. This strategy improves upon conventional yeastdisplay capture by multiplying the number of protein binding domainsthereby increasing metal capture capacity up to 1-10 mM. The secondstrategy focuses on metal internalization by engineering metal transportsystems at the cell membrane and vacuole. Using engineered SMF1 membranetransporter and CCC1 vacuole transporter, uptake of cadmium increases10-fold when compared to WT. In certain embodiments, SMF1 can be beingfurther modified to become sensitive to other relevant metals such asstrontium, lead, and mercury. Finally, the third strategy uses hydrogensulfide by-products from the yeast sulfate-assimilation pathway to reactand precipitate heavy metals from solution. In certain embodiments, theformation of metal sulfide particles can be controlled with yeastdisplayed biomineralization peptides, or modulating hydrogen sulfideproduction rate, in order to obtain useful metal sulfide nanoparticlessuch as quantum dots. Strategies 1-3 can be combined to provide apowerful method for heavy metal remediation. Current ideas tosynergistically combine the various strategies of metal binding,transport, and mineralization are shown in FIGS. 20A-20C.

These strategies can be used to build a platform in which industries andthe public can accessibly and cheaply purify water. What makes yeastsuch an attractive platform is the ease to engineer and rapidly testbetter performing strains, and this can only get simpler with moreadvanced genetic engineering tools. In addition yeast circumvents manylimitations hampering current physicochemical processes, such asrenewability, cost, and production of secondary waste. Already theindustry to cheaply scale the production of yeast exists because of thebeer and pharmaceutical industry. See Argyro Bekatorou, CostasPsarianos, and Athanasios A Koutinas. Production of food grade yeasts.Food Technology and Biotechnology, 44(3):407-415, 2006, which isincorporated by reference in its entirety. Likewise, the food industryhas establish protocols to handle, transport, and store yeast forconsumer use, so there exist a feasible entry way for this technology toenter the public market. The methods disclosed here can be combined withalready established yeast production infrastructures in order to provideyeast at low costs. Current infrastructures from the beer, pharma, andfood industries can be utilized to create another avenue in which yeastcan remediate toxic materials from industrial processes such as mining,chemical spills, and manufacturing runoff.

Examples

Yeast as a Sequestration Agent

A yeast strain that endogenously displays the AGA1 & AGA2 surfaceproteins was designed by constructing a DNA construct containing theAGA1 and AGA2 sequence with a strong constitutive promoter (GPO) and acanonical transcription terminator (CYC1). In addition, the AGA2 isfollowed by a protein of interest (POI) flanked by restriction sitesNheI and XhoI (for cloning purposes) and a N′—HA and C′-Flag tag (forexpression studies). Finally, the construct is appended to a TRP1autotrophic marker for positive selection of transformed yeast.

The POI are a family of plant metallothioneins (MT1A-4A) and the yeastendogenous metallothionein (CUP1). Metallothioneins are known to havestrong affinities for copper, zinc, mercury, and lead. See Robinson,Nigel J., et al. “Plant metallothioneins,” Biochemical Journal 295.Pt 1(1993): 1. Yeast display of the AGAI & AGA2 constructs is used toexpress multiple copies of these metallothioneins (50,000-100,000copies; see Boder. Eric T., and K. Dane Wittrup, “Yeast surface displayfor screening combinatorial polypeptide libraries,” Nature biotechnology15.6 (1997): 553-557, which is incorporated by reference in itsentirety) on a single yeast surface to act as a metal binding domains.

TABLE 1 Table listing the 4 families of metallothioneinsfrom Arabidopsis thaliana and the yeastmetallothionein (CUP1) used for yeast display SEQ ID MT geneProtein Sequence NO MT1A MADSNCGCGSSCKCGDSCSCEKNYNKECDNCS 4CGSNCSCGSNCNC MT2A MSCCGGNCGCGSGCKCGNGCGGCKMYPDLGFS 5GETTTTETFVLGVAPAMKNQYEASGESNNA ENDACKCGSDCKCDPCTCK MT3MSSNCGSCDCADKTQCVKKGTSYTFDIVETQE 6 SYKEAMIMDVGAEENNANCKCKCGSSCSCVNCTCCPN MT4A MADTGKGSSVAGCNDSCGCPSPCPGGNSCRCR 7MREASAGDQGHMVCPCGEHCGCNPCNCPKT QTQTSAKGCTCGEGCTCASCAT CUP1NIFSELINFQNEGHECQCQCGSCKNNEQCQKSC 8 SCPTGCNSDDKCPCGNKSEETKKSCCSGK

Data presented in this report are with respects to plant Arabidopsisthaliana metallothionein MT2A for conciseness and because all MTs testedshow extremely similar results. Yeast display expression of MT2Aincreases Cu(II) uptake by 4-5 fold compared to WT which is >100 timeshigher than the US Environmental Protection Agency (EPA) actionablelevel of 1.3 ppm for allowable copper concentrations in drinking water.Seefile:///C:/Users/GeorgeSun/AppData/Roaroing/Zotero/Zotero/Profiles/wc3qz9ge.default/zotero/storage/S.regulated-drinking-water-contaminants.html,which is incorporated by reference in its entirety. In addition, MT2Ayeast display strains are able to tolerate and thrive in high copperconcentrations of about 16-20 mM, whereas WT die below 5 mM.

The plant metallothionein protein can be further engineered or evolvedand screened for greater metal binding efficiency, capacity, and/orselectivity via high-throughput genetic engineering and screeningmethods such as flow cytometry.

Yeast as a Metal Absorber

At a different perspective, the yeast's entire intracellular volume canbe viewed as a sequestration locale. Several yeast strains thatover-express a unique metal transporter (Table 2) were created byfollowing similar DNA design strategies as described above (FIG. 21).See, Hall, J. L., and Lorraine E. Williams, “Transition metaltransporters in plants,” Journal of experimental botany 54.393 (2003):2601-2613, which is incorporated by reference in its entirety. Eachmetal transporter gene is controlled by a GAL1 inducible promoter andfollowed by a V5 epitope tag (for staining purposes) and a CYC1transcription terminator.

Of the expressed strains, metal uptake for Cu(II), Zn(II), and Mn(II)were tested.

TABLE 2 A list of most prominent metal transporters involved in yeast.Name Description ZRT1 High-affinity zinc transport protein ZRT2Low-affinity zinc transport protein ZRT3 Transports zinc from storage inthe vacuole to the cytoplasm ZAP1 Involved in zinc ion homeostasis byzinc•responsive transcriptional regulation CTR1 Required for highaffinity copper (probably reduced Cul) transport into the cell CTR2Provides bioavailable copper via mobilization of vacuolar copper storesand export to the cytoplasm CTR3 Required fur high affinity copper(probably reduced Cul) transport into the cell FRE1 Metalloreductaseresponsible for Fe3+ and Cu2+ salts FRE2 Metalloreductase responsiblefor Fe3+ and Cu2+ salts FTR1 Permease for high affinity iron uptake FET3Iron transport multicopper ferroxidase required for Fe2+ ion highaffinity uptake. Required to oxidize Fe2+ and release it from thetransporter. Essential component of copper-dependent iron transport FET4Required for Fe2+ ion low affinity uptake BSD2 Required for homeostasisof heavy metal ions such as cadmium, cobalt and copper. Undermanganese-replete conditions facilitates trafficking of SMF1 and SMF2metal transporters to the vacuole where they are degraded SMF1High-affinity manganese transporter involved in manganese uptake fromthe extracellular environment. Contributes also to cellular accumulationof other divalent metal ions such as cadmium, cobalt, copper, iron andnickel SMF2 High-affinity manganese transporter involved in mobilizingmanganese from vesicular stores in conditions of low manganese ionconcentrationsRecycling & Conversion of Toxic Metals Using Yeast Display

A metal's toxicity is based on its oxidation state and the molecularcompound in which it is in. For example, Cr(VI), specifically chromates(CrO₄ ²⁻, Cr₂O₇ ²⁻) is considered highly mutagenic and acutely toxic,however Cr(III) is insoluble in water and is overall less reactive.Therefore, it is equally important to capture heavy metals as it is toconvert and possibly recycle these metals to a more benign and usableform.

Organisms have already discovered methods to convert metals from anunfavorable state to a more favorable one. Specifically, cytochromesfound in dissimilatory metal reducing bacteria are known to transferelectrons to heavy metals as terminal electron accepters in order togenerate an electrochemical gradient for the production of chemicalenergy. See Lovley, Derek R. “Dissimilatory metal reduction.” AnnualReviews in Microbiology 47.1 (1993): 263-290, which is incorporated byreference in its entirety. These proteins all contain a porphyrincofactor in which a chelated metal facilitates the transfer of electronsfrom a metabolic substrate (i.e. conversion of NAD(P)H, formate,lactate, or pyruvate, etc.) to heavy metals from the environment. Asimilar mechanism is being pursued in yeast in which yeast cytochromes,CYC1 & 7, are displayed on the surface in hopes of facilitating electrontransfer between environmental heavy metal and the cytochrome's hemegroup. An increase in redox potential of yeast displayed CYC1/7 wasobserved as evidence for the possibility of facilitated heavy metalreduction.

Biologically catalyzed metal reduction can be further pursued to createmicrobial fuel cells in which an organism, typically bacterial catalyzesthe oxidation or reduction of an organic or inorganic matter to generatecurrent. The same principal to reduce metals using yeast displayedcytochromes can simultaneously be harnessed to generate and storecurrent ill a fuel cell.

Metal-Contaminated Yeast after Water Treatment

Metal-contaminated yeast filter packets can be handled many differentways after water treatment. The first is to reuse or recycle the filterpacket by removing the captured heavy metal. The most direct method torelease captured metals is to gently wash the yeast filter packet formetal removal at reduced pH (pH 3-4). The second method is toenzymatically treat the yeast with proteases to cleave the protein-metalcomplexes off the cells. After separation, yeast can be air-dried,stored, and later reconstituted when another batch needs to be cultured(FIG. 25). In FIG. 25, the first stage (green layer) provides an easymethod to culture and propagate stocks of yeast. Yeast can be grownindefinitely given enough space and nutrients, or stored andconsolidated in packets for later use. When yeast are needed (orangelayer), cultures can be aliquoted from the main stock and added into theremediation filter packet. Contaminated waters can be manually pumped orwashed over the filter packet to capture any metal contaminants. Fromhere, yeast can be separated from the captured metal by washing atreduced pH, enzymatic, or salt treatment (blue layer). Yeast can bereused, composted, or autolyzed as a nutrient source for subsequentculture growth. Captured metals should be thrown out to dedicated wastesites or delivered to industries or government agencies that can recyclethe metals.

A drop in strain integrity was observed after remediation, such asslower growth rates, incomplete separation of contaminants, or strainmutations, yeast can be lysed and then biodegraded. Lysis simplyrequires salted water, approximately a few hundred millimolar (6-30grams per liter of water) (see Huh, G.-H. et al. Salt causes iondisequilibrium-induced programmed cell death in yeast and plants. PlantJ. 29, 649-659 (2002), which is incorporated by reference in itsentirety), to create a hypertonic environment in which yeast undergoautolysis. Hypertonic solutions swell and eventual burst the yeast cellreleasing enzymes and proteases causing cellular degradation. Autolysiseffectively reduces yeast biomass into yeast extract, a rich blend ofbasic nutrients such as amino acids and nitrogen sources that can feedfuture cultures of yeast. See Chae, H. J., Joo, H. & In, M.-J.Utilization of brewer's yeast cells for the production of food-gradeyeast extract. Part 1: effects of different enzymatic treatments onsolid and protein recovery and flavor characteristics. Bioresour.Technol. 76, 253-258 (2001), and Tanguler, H. & Erten, H. Utilisation ofspent brewer's yeast for yeast extract production by autolysis: Theeffect of temperature. Food Bioprod. Process. 86, 317-321 (2008), eachof which is incorporated by reference in its entirety. Yeast extract isnot toxic (as yeast extract is commonly used as a food additive forflavoring). There also exist commercial food brands based on yeastextract such as Vegemite and Marmite.

Target Heavy Metals

Two categories of heavy metals can be considered. The first category isdivalent elemental metals such as lead (Pd²⁺), mercury (Hg²⁺), cadmium(Cd²⁺), etc. The second category is polyatomic and organic metals suchas chromium, which is typically found as chromate (CrO₄ ²⁻); arsenic,which is typically found as arsenate (AsO₄ ³⁻); and organomercury, whichis found in numerous states with methyl functional groups (e.g.,CH₃—Hg⁺).

The first category was successfully demonstrated in terms of enhancinguptake capacity, specificity, and ability to sequester andcompartmentalize elemental metal contaminants. However, for polyatomicand organic metal compounds, due to their different chemistries,differing yeast metabolic pathways, and obviously the difference incharge and valency, the strategies to address elemental metal captureneed to be adjusted. Yeast permeases, in particular sulfate (SO₄ ²⁻) andphosphate (PO₄ ³⁻) permeases, can be used to uptake chromate (CrO₄ ²⁻)and arsenate (AsO₄ ³⁻), respectively, since the structural similaritybetween the two species would allow for direct pumping in of the metaloxide counterpart. Hyperaccumulation of chromate and arsenate using twosulfate permease genes (Sul1 and Sul2) and are in a position to furtherengineer and optimize the system. The other two strategies, yeastsurface capture and mineralization are still being engineered toaccommodate this second category of targets. This technology can be usedfor water treatment in areas such as Flint as well as other neglectedareas poisoned by industrial runoff and mining effluent.

Safety for Drinking Water Applications

An easy to follow and robust method for water treatment that isadaptable to geographical location, types of water sources, user levelof expertise, in addition to withstanding common user modes of failurecan be provided. The mode of operation is to filter water through ayeast packet and funnel the flow through into a water container.Alternatively, the packet can be submerged in the treated water (muchlike a sponge) where the water can then be collected.

To ensure robustness of yeast filter packet, treated waters can bestrained in a size-exclusion filter to remove particulate and yeast.Commonly used and available size-exclusion filters with 0.2-0.5 μm (seeCorning sterile filtration guide, available at:www.corning.com/media/worldwide/cls/documents/CLS-FIL-004%2OREV4%20DL.pdf (accessed: 30 Mar. 2017), which is incorporated by reference in itsentirety) pore sizes are small enough to segregate yeast from enteringthe water.

Also, the strains are engineered to be autotrophic, that is they lackthe capacity to produce essential nutrients for growth, typically aminoacids such as tryptophan, histidine, leucine etc. Unless supplied by theuser (during culture), yeast cannot survive and will eventually die ifoutside of filter.

The engineered strains are able to flocculate given an externalstimulus. The packet can contain factors that suppress flocculation. Inthe event that the yeast reside outside the filter, then without accessto the flocculation suppressing factors, flocculation would occur, whichinduces yeast precipitation and automatic removal from the water.

A variety of pH- and metal-tolerant biodegradable hydrogels canencapsulate and secure yeast in a contained filter packet. These packetscan then be surrounded in a semi-porous membrane (e.g. similar todialysis tubing) to allow diffusion of treated water into the packet,yet disallow movement of larger molecules beyond a given molecular sizecutoff. See Ma, Y. et al. Effects of nanoplastics and microplastics ontoxicity, bioaccumulation, and environmental fate of phenanthrene infresh water. Environ. Pollut. 219, 166-173 (2016), Gimpel, J., Zhang,H., Davison, W. & Edwards, A. C. In Situ Trace Metal Speciation in LakeSurface Waters Using DGT, Dialysis, and Filtration. Environ. Sci.Technol. 37, 138-146 (2003), and Nolan, A. L., Mclaughlin, M. J. &Mason, S. D. Chemical Speciation of Zn, Cd, Cu, and Pb in Pore Waters ofAgricultural and Contaminated Soils Using Donnan Dialysis. Environ. Sci.Technol. 37, 90-98 (2003), each of which is incorporated by reference inits entirety. The hydrogels in the packet are meant to be degraded sothat metals can be stripped and segregated from the yeast after use.Yeast can then be harvested for re-inoculation or degradation asdescribed in FIG. 25.

If the packet degrades after successive water treatments, thesemi-porous membrane adds another barrier of selection against particlesdissolving into the water. The dialysis tubing, most likely made ofnitrocellulose or a cellulose monomer, is typically resistant to manychemicals and pH ranges and has long shelf and usage lifetimes. SeeSnakeSkin Dialysis Tubing, available at:tools.thermofisher.com/content/sfs/manuals/MAN0011339_SnakeSkin_Dialy_Tubing_UG.pdf(accessed: 30 Mar. 2017), and Spectra Cellulose Dialysis Membrane,available at: www.spectrumlabs.com/lit/420x10116x000.pdf (accessed: 30Mar. 2017), each of which is incorporated by reference in its entirety.

Although yeast has been engineered to secrete a variety of proteins forcommercial and pharmaceutical purposes, native yeast has a limitedsecretome. See Choi, J. et al. Fungal Secretome Database: Integratedplatform for annotation of fungal secretomes. BMC Genomics 11, 105(2010), which is incorporated by reference in its entirety. Mostsecreted proteins are typically mating factors or pheromones needed tocommunicate with other cells during haploid mating events. See Loumaye,E., Thorner, J. & Catt, K. J. Yeast mating pheromone activates mammaliangonadotrophs: evolutionary conservation of a reproductive hormone?Science 218, 1323-1325 (1982), which is incorporated by reference in itsentirety. If this is any cause for concern, these pathways can be easilyknocked out to limit the amount of factors secreted. Secretion may notbe a concern given that beer is essentially the collection and reductionof yeast supernatant (these strains will not be fermented, so noproduction of sugars or alcohols should exist in the filtered water).

To prevent incomplete metal capture, preliminary on-site measurementscan be performed to see how this yeast filter packet capture capacityscales with the amount of metal contaminants in the local waters to betreated. Given an approximate estimate of local contaminantconcentrations, different sizes or densities of yeast packets can beused to efficiently remove all contaminants in one treatment cycle.Alternatively, multiple yeast packets can be used in-tandem pertreatment for increased capture capacity.

In the treatment pipeline, a metal sensing packet can be introduced. Apacket containing yeast can colorimetrically or fluorescently respond tometal contaminants. For example, the packet can contain yeast that has agreen fluorescent protein (GFP) downstream of a metal-inducible promoter(FIG. 26). These packets can be separated from the remediation packet,but can still follow the culturing and usage pipeline outlined in FIG.25. Promoters include those that control metallothionein and glutathionetranscription (CUP1, MTF-1, etc.), which can be used to control GFPexpression. See Saydam, N., Adams, T. K., Steiner, F., Schaffner, W. &Freedman, J. H. Regulation of Metallothionein Transcription by theMetal-responsive Transcription Factor MTF-1 IDENTIFICATION OF SIGNALTRANSDUCTION CASCADES THAT CONTROL METAL-INDUCIBLE TRANSCRIPTION. J.Biol. Chem. 277, 20438-20445 (2002), and Ecker, D. J. et al. Yeastmetallothionein function in metal ion detoxification. J. Biol. Chem.261, 16895-16900 (1986), each of which is incorporated by reference inits entirety.

Wastewater Remediation Versus Drinking Water

Wastewater remediation is of great interest, particularly mining andagricultural runoff.

However, the most pressing issue in developing areas and economicallydisadvantaged communities is the inaccessibility of safe drinking water.The disclosed method can provide a renewable platform to continuouslygrow and use water remediation agents (yeast) that can empowercommunities to clean their own waters. Accessible and usable in DIYpackets with yeast can be grown and maintained in a user-friendly mannerin many geographical regions.

Yeast Versus Sulfate-Generating Bacteria (to Precipitate MetalloidSulfides)

Yeasts are easier to grow, have shorter doubling times, and have anextensive toolkit for molecular biology engineering. In addition, yeaststrains that are able to produce sulfur require the same nutrients aswild-type strains, as the deletion of the sulfate-assimilation pathwaydoes not perturb any other metabolic pathway.

In comparison to sulfur-generating bacteria, commonly found in thefamilies of Desulfobacterales, Desulfovibrionales andSyntrophobacterales (see Muyzer, G. & Stams, A. J. M. The ecology andbiotechnology of sulphate-reducing bacteria. Nat. Rev. Microbiol. 6,441-454 (2008), which is incorporated by reference in its entirety),these strains are very difficult to grow and engineer compared to yeast.These cultures require anaerobic conditions, meaning oxygen is lethal totheir growth and require controlled anaerobic chambers devoid of oxygenwith precise control of humidity and temperature. In addition,production of sulfur requires additional nutrients that are otherwisenot needed for commonly used bacteria and yeast strains, such aspropionate, high sulfate concentrations, or fermentable lactate andethanol. See Muyzer, G. & Stams, A. J. M. The ecology and biotechnologyof sulphate-reducing bacteria. Nat. Rev. Microbiol. 6, 441-454 (2008),and H Kadota & Ishida, and Y. Production of Volatile Sulfur Compounds byMicroorganisms. Annu. Rev. Microbiol. 26, 127-138 (1972), each of whichis incorporated by reference in its entirety. Even under the best growthconditions, the pathway and control of sulfur production insulfur-generating bacteria are relatively unclear and still requirefurther investigation. See Schippers, A. & Sand, W. Bacterial Leachingof Metal Sulfides Proceeds by Two Indirect Mechanisms via Thiosulfate orvia Polysulfides and Sulfur. Appl. Environ. Microbiol. 65, 319-321(1999), and Friedrich, C. G., Rother, D., Bardischewsky, F., Quentmeier,A. & Fischer, J. Oxidation of Reduced Inorganic Sulfur Compounds byBacteria: Emergence of a Common Mechanism? Appl. Environ. Microbiol. 67,2873-2882 (2001), each of which is incorporated by reference in itsentirety. Understanding this pathway is further hindered by thedifficulty of genetically engineering sulfur-generating strains, as theyoffer few avenues for genetic manipulation compared to more evolvedbacteria and yeast strains. Strain engineering in bacteria usuallyrequires horizontal gene transfer of a shuttle vector from awell-defined and engineerable host such as bacteria; this added layer ofcomplexity overall slows the engineering pipeline for improved strainperformance. See Dodsworth, J. A. et al. Interdomain Conjugal Transferof DNA from Bacteria to Archaea. Appl. Environ. Microbiol. 76, 5644-5647(2010), which is incorporated by reference in its entirety.

To summarize, the engineered yeast strains require minimal cultureeffort, grow at ambient temperature with limited impact on ambient airconditions, and produce appreciable quantities of sulfur. In ahead-to-head comparison with respect to sulfur production, theengineered strains can produce 55±8 ppm of sulfur per culture (12 hoursof growth in an Erlenmeyer flask), whereas depending on the bacteriastrain, nutrient source, and culturing method (flask, fermentationchamber, bioreactor, etc.) production of sulfur can range from 7.5 to 67ppm (see Jong, T. & Parry, D. L. Removal of sulfate and heavy metals bysulfate reducing bacteria in short-term bench scale upflow anaerobicpacked bed reactor runs. Water Res. 37, 3379-3389 (2003), which isincorporated by reference in its entirety) in 12 hours. Yeast is favoredbecause of its ease of use, known sulfur pathway, and controllability ofsulfur production.

Scalability and Preliminary Cost Analysis of DIY Yeast Packets

Beer industry can be used as a reference on the economics of yeastproduction and cite academic literature on bioseparation processes anddistribution for evaluating the scalability and cost of the DIY yeastpackets. The United States alone produced 55 billion gallons of beer in2012. These yeasts are either recycled for use in another productionbatch or discarded. These numbers do not consider the production ofyeast for consumer and pharmaceutical goods like bread, dried-yeastpackets, and therapeutic compounds which is on the same order ofmagnitude as the 55 billion gallons of beer in 2012. See The Economicsof Craft Beer|SmartAsset.com. SmartAsset (2017), available at:smartasset.com/credit-cards/the-economics-of-craft-beer (accessed: 2Apr. 2017), which is incorporated by reference in its entirety.Typically, 2-10 billion yeast cells are needed to ferment a singlegallon of beer (see Baker, D. A. & Kirsop, B. H. Rapid Beer Productionand Conditioning Using a Plug Fermentor. J. Inst. Brew. 79,487-494(1973), which is incorporated by reference in its entirety), makingyeast production estimated at approximately 24 thousand tons of biomassper year (see Bryan, A. K., Goranov, A., Amon, A. & Manalis, S. R.Measurement of mass, density, and volume during the cell cycle of yeast,Proc. Natl. Acad. Sci. 107, 999-1004 (2010), which is incorporated byreference in its entirety). What this means is that yeast is already acheap, scalable, and consumer friendly type of microorganism that can besimilarly scaled and distributed for water remediation purposes.

In typical bioprocessing settings (averaged for pharmaceuticalapplications) the cost of raw yeast is approximately $4 dollars perkilogram. See Harrison, R. G., Todd, P., Todd, P. W., Rudge, S. R. &Petrides, D. P. Bioseparations Science and Engineering. (OxfordUniversity Press, 2015), which is incorporated by reference in itsentirety. Ingredients to maintain cultures such as glucose, yeastextract, amino acids, and trace elements go for $3 dollars per kilogramof total material. Id. In the lab, typically 1 million cells are seededper mL of yeast in 1 liter cultures. Therefore, the cost to start aculture is 16 cents per L culture. In the laboratory setting, andespecially in batch and fermentation processes, yeast can undergoseveral doublings per inoculum. A final 1-liter culture may have as muchas 1¹⁰-1¹¹ cells in 16 hours (experimentally determined). Therefore, thefinal cost of yeast per cost of raw material is most likely an order ofmagnitude lower than what is calculated.

With respects to the cost of yeast needed per packet, assumingcontamination levels of up to 100 μM or more (equivalent to ten to athousand times higher than EPA standards for certain metals; see EPA.Wastewater Technology Fact Sheet Chemical Precipitation (2000),available at:nepis.epa.gov/Exe/ZyPDF.cgi/P1001QTR.PDF?Dockey=P1001QTR.PDF (accessed:5 Jan. 2017), which is incorporated by reference in its entirety), 1¹⁰yeast cells or less would be required to completely purify a llitersolution (based on experimental results). Therefore, a packet containing1¹⁰ cells would require a fraction of a liter, meaning the cost topurify water can start as low as a few pennies per liter of drinkingwater.

Therefore, the most expensive aspect of setting up a culture is the costof equipment, transportation, and packaging. Breaking down the costfurther, a pound (454 g) of freeze- or active-dried yeast for DIYbrewing or bread making costs 5-30 dollars depending on the quality ofyeast (i.e. number of surviving cells, QC testing, brand identification,etc.). Home brewing kits (typically sized as 5 gallons) can go for $30to $300 dollars, again depending on the quality of the brand. SeeAssociation, H. A. Is Homebrewing Cheaper than Store-Bought Beer?American Homebrewers Association (2015), available at:www.homebrewersassociation.org/news/is-homebrewing-cheaper-than-store-bought-beer/(accessed:2 Apr. 2017), which is incorporated by reference in its entirety.Therefore, to culture 1 L of yeast costs roughly $5-10 dollars on thelow end. However, the startup cost can be lowered by using standardculturing flasks, simple ingredients, and the ability to freeze-dryone's own cultures to self-propagate cultures for future use.

Large-Scale Comparison of DIY Yeast Packets Versus PhysicochemicalMethods

Whether synthesizing resins, adsorption filters, or electrochemicalsubstrates, the basic processing pipeline is as follows (see Harrison,R. G., Todd, P., Todd, P. W., Rudge, S. R. & Petrides, D. P.Bioseparations Science and Engineering. (Oxford University Press, 2015),and Jr, W. D. C. & Rethwisch, D. G. Fundamentals of Materials Scienceand Engineering: An Integrated Approach. (John Wiley & Sons, 2012), eachof which is incorporated by reference in its entirety): (1) reactors andsynthesis, Primary recovery—solid and phase separation, (2) intermediaterecovery—ultrafiltration, evaporation, reverse osmosis, etc., (3) finalpurification—crystallization, solvent/pH exchange, chromatography, etc.,(4) quality control (QC)—size, structure, chemicalcomposition/characterization, etc., and (5) packaging andstorage—typically requires technical handling and storage. Whereasbioprocessing of yeast requires: (1) continuous bioreactor, (2) cellseparation—filtration or centrifugation of cellular debris, (3)QC—routine genotyping, and (4) packaging and storage—freeze-dried,air-dried, etc.

Yeast has advantages as it avoids complex synthesis steps, requires asimple isolation process, and QC reduces to simple strain genotyping toguarantee selection of engineered yeast. Moreover, iteration becomesmore feasible as molecular biology engineering requires simple cloningtechnologies, such as DNA oligo design, polymerase chain reaction (PCR),and transformations, which can cost as little as a few dollars perexperiment. See Guthrie, C. & Fink, G. R. Guide to yeast genetics andmolecular and cell Biology: Part C. 351, (Gulf Professional Publishing,2002), which is incorporated by reference in its entirety. Forphysicochemical processes, however, an entire synthesis pipeline mayneed to be adjusted to accommodate changing reaction conditions, andpossibly entire facilities may need to be retro-fitted in case ofincompatible reaction steps.

When compared with the current resin technology (chromatography), resinsrequire chemical functionalization of polystyrene beads whosechemistries can be quite complex, making optimization of highlyspecified and selective resin groups difficult and slow. See KentaroTashiro, Modular synthesis of metal-organic complex arrays containingprecisely designed metal sequences, available at:globalscience.berkeley.edu/sites/default/files/15-modularmoca.pdf(accessed: 2 Apr. 2017), which is incorporated by reference in itsentirety. Likewise, resin-based technology may not be amenable forpublic use as storage conditions may differ per resin type andfunctional group, and resins are not easily recycled if users do nothave technical knowledge of the precise chemistries and best practicesfor handling. Therefore, resins may actually contribute to secondarywaste if not properly managed.

On the other hand, yeast provides a better alternative for resin watertreatment, especially in the form of yeast display. Surface proteinexpression is easily and greatly tunable on the genetic level whilemaintaining identical culturing conditions for un-engineered, or otherengineered yeast strains. Likewise, yeast can be re-used or stored forlater use as described in the sections above. But if yeast were to bediscarded, they are biodegradable and would not contribute to secondarywaste.

Compared to the current adsorption filters, similar arguments with resintechnology, the manufacturing and chemistries of adsorption filters andmembranes inhibit this technology from being widely distributed forpublic use. The first is the decline, or scarcity of resource materialssuch as nanostructured materials or carbon nanotubes. See Jong, T. &Parry, D. L. Removal of sulfate and heavy metals by sulfate reducingbacteria in short-term bench scale upflow anaerobic packed bed reactorruns. Water Res. 37, 3379-3389 (2003), and Stafiej, A. & Pyrzynska, K.Adsorption of heavy metal ions with carbon nanotubes. Sep. Purif.Technol. 58, 49-52 (2007), each of which is incorporated by reference inits entirety. Likewise handling and recycling of adsorption filters andmembranes may be difficult to manage for untrained users.

One of the most highly used physicochemical methods for industrial wastetreatment, chemical precipitation uses sacrificial iron compounds orreactive hydroxyl or sulfur groups to precipitate and remove metalcomplexes. See Charerntanyarak, L. Heavy metals removal by chemicalcoagulation and precipitation. Water Sci. Technol. 39, 135-138 (1999),which is incorporated by reference in its entirety. For scaledindustrial use chemical precipitation costs are relatively cheap:approximately $0.05-$0.2 per liter of water in the US. See Ozturk, I.,Altinbas, M., Koyuncu, I., Arikan, O. & Gomec-Yangin, C. Advancedphysico-chemical treatment experiences on young municipal landfillleachates. Waste Manag. 23, 441-446 (2003), which is incorporated byreference in its entirety. However, this comes with hazardous pH rangesof 10-12 and handling of several grams of chemicals per liter.Additionally, sulfur is becoming a more prominent player in chemicalprecipitation for its speed and reactivity; however, chemical storage ofsulfide in the form of sodium sulfide, iron sulfide, or sulfuric acidprecursor is incredibly dangerous to handle and should be avoided inpublic hands. See Charerntanyarak, L. Heavy metals removal by chemicalcoagulation and precipitation. Water Sci. Technol. 39, 135-138 (1999),which is incorporated by reference in its entirety.

The production of hydrogen sulfide from a biological source is morebenign than direct chemical precipitation. There is no need for storageof precursor chemicals for sulfide production, as the culture alreadycontains the nutrients needed for yeast to metabolize. So if sulfurproduction were to be controlled, yeast can be easily moved from themedia and idled in another buffer source or stored for future use.Finally, the sulfur content remains in solution and those that do becomevolatile and evaporate into the atmosphere where the concentrationdramatically reduces to safe levels below EPA standards and is not aprominent safety concern. See EPA. Public Health Statement—HydrogenSulfide, available at: www.atsdr.cdc.gov/toxprofiles/tp114-c1-b.pdf(accessed: 2 Apr. 2017), and By Scott Simonton, P. D. & Oct. 3, 2007.Human Health Effects from Exposure to Low-Level Concentrations ofHydrogen Sulfide. Occupational Health & Safety, available at:ohsonline.com/articles/2007/10/human-health-effects-from-exposure-to-lowlevel-concentrations-of-hydrogen-sulfide.aspx(accessed: 2 Apr. 2017), each of which is incorporated by reference inits entirety.

Regionally Specific, Personally Customized Deployable DIY Yeast Packetsfor Heavy Metal Remediation from Contaminated Water

The engineered yeast can be packaged into user-friendly and economicalunits that can be deployed in areas in need of heavy metal remediationto provide strains of yeast that are regionally tailored so that userscan simply grow and maintain their own batches of yeast for personalwater remediation efforts. Instructions along with an ease to useculture containers can provide users a means to regularly grow their ownyeast stocks for routine and self-sufficient water purification.

Yeast cell surface display of plant-based metal-binding proteins, knownas metallothioneins (MTs), is analogous to the physicochemicalion-exchange technique. Four families of plant MTs (MT1A-MT4A in FIG.27B) as well as the yeast endogenous metallothionein (CUP1) wereexpressed on the yeast surface and assayed for Cu²⁺ uptake (the nativeligand preferred by the metallothionein families) (FIG. 27A). The AGA1and AGA2 (purple) domains were used to express peptides or proteins withmetal binding domains to remove contaminants from waters (orangecircles). Expression of MTs increased uptake capacity of Cu²⁺ by 3-4fold compared to wild-type yeast (FIG. 27B). In addition, expression ofMTs increased tolerance of copper concentration in solution by 3-4 fold(FIG. 27C).

Given modest estimates of yeast density and expression levels, the upperlimit of yeast display capture is in the submillimole range per gram ofdry weight. On the other hand, synthetic ion-exchange resin capacitiesare in the 10 μmol-10 mmol of metal per gram of material (see Barakat,M. A., New Trends in Removing Heavy Metals from Industrial Wastewater.Arabian Journal of Chemistry 2011, 4 (4), 361-377, and Stathi, P.;Papadas, I. T.; Tselepidou, A.; Deligiannakis, Y., Heavy-Metal Uptake bya High Cation-Exchange-Capacity Montmorillonite: The Role of PermanentCharge Sites. Global NEST Journal 2010, 12 (3), 248-255, each of whichis incorporated by reference in its entirety), up to 1-3 orders ofmagnitude greater than that demonstrated with the metallothionein yeastdisplay experiments (FIG. 27B). Despite an overall lower bindingcapacity, an advantage of using yeast display as a method for metalcapture is the ability to engineer highly specific metal-binding domainsfor extremely toxic metals, such as mercury, while avoiding nonspecificsaturation from common ions such as Na, Ca, Mg, etc.

The shortcomings of traditional yeast cell surface display can beovercome, while retaining the ability to engineer heavy metalspecificity, by the use of externally secreted multiplier proteins,which greatly increase the number of metal-binding domains available pergram of dry weight. MTs were then fused to a multiplier protein,glutamine synthetase (GS). GS is a dodecahedron protein with 12subunits. Each subunit can be fused with a MT appendage on theN′-terminus. Glutamine synthetase was engineered to aggregate inresponse to a range of metals from zinc to cadmium (FIG. 2). This systemuses one strain to express a single copy of the GS subunit on the yeastsurface as the anchorage protein, with another strain secreting theGS-MT complex. In the presence of metals secreted GS-MT complexes beingto aggregate and form on the yeast surface producing a mesh network(FIG. 4). Binding capacities are estimated to be enhanced by 2-3 ordersof magnitude given the increased amount of metal uptake than compared tosingly displayed MTs.

The plausibility of using yeast as a platform for metal remediationbecomes even more readily apparent when incorporating multiplierproteins and proteins engineered for metal-specific binding into theremediation strategy. Doing so increases capture capacities to 1-10 mM,on par with synthetic ion-exchange resin capacities, and allows forspecific metal uptake (FIG. 5, lower). In addition, due to their greaterdensity, yeast bound to metals can settle out of solution allowing foreasy physical separation from remediated waters (FIG. 5, upper).

In addition to possessing metal-binding proteins (MTs), certain plantscan uptake large quantities of metals, known as hyperaccumulation. SeeClemens, S.; Palmgren, M. G.; Krämer, U., A Long Way Ahead:Understanding and Engineering Plant Metal Accumulation. Trends in PlantScience 2002, 7 (7), 309-315, which is incorporated by reference in itsentirety. Such hyperaccumulators often use metal transporter proteinsfor subsequent compartmentalization into organelles called vacuoles orbind metals using sequestration agents, primarily in the form ofphytochelatins or metallothionein proteins. See Song, W. Y.; Park, J.;Mendoza-Cozatl, D. G.; Suter-Grotemeyer, M.; Shim, D.; Hortensteiner,S.; Geisler, M.; Weder, B.; Rea, P. A.; Rentsch, D.; Schroeder, J. I.;Lee, Y.; Martinoia, E., Arsenic Tolerance in Arabidopsis Is Mediated byTwo Abcc-Type Phytochelatin Transporters. Proceedings of the NationalAcademy of Sciences of the U.S. Pat. No. 2,010,107 (49), 21187-21192,and Cobbett, C.; Goldsbrough, P., Phytochelatins and Metallothioneins:Roles in Heavy Metal Detoxification and Homeostasis. Annual Review ofPlant Biology 2002, 53, 159-182, each of which is incorporated byreference in its entirety. However, because plants are stationary,difficult to biologically engineer, and have a long growth cycle, plantsare not the best candidates to develop a modular platform for a range ofmetal remediation tactics. Instead, this strategy is to use plants asinspiration to engineer yeast for heavy metal uptake andcompartmentalization. There already exist numerous yeast metaltransporters similar to those of plants that respond to variousconditions such as pH, cofactors, and/or energy resources. The same isalso true for vacuole transporters that secure toxins away from theyeast body.

Difficulty in predicting metal-binding regions and metal specificityregions in these transporter proteins has previously prevented rationaldesign to attain better performance. See Arguello, J. M., Identificationof Ion-Selectivity Determinants in Heavy-Metal Transport P-1b-TypeATPases. Journal of Membrane Biology 2003, 195 (2), 93-108, which isincorporated by reference in its entirety. Here, a screening method canuse density changes in the yeast cell as a direct measurement toqualitatively discern metal uptake efficiency. Using established valuesfor yeast cell density and mass (see Bryan, A. K.; Goranov, A.; Amon,A.; Manalis, S. R., Measurement of Mass, Density, and Volume During theCell Cycle of Yeast. Proceedings of the National Academy of Sciences ofthe United States of America 2010, 107 (3), 999-1004, which isincorporated by reference in its entirety), even metal uptake in thehundreds of μM can induce density changes up to 25% depending on themolar mass of the metal. Density changes can be discerned using densitygradient separation techniques, such as Percoll density centrifugation,which has a density resolution on the order of 2-3%. See Ravnik, S. E.;Gage, S.; Pollack, S. B., Self-Generating Density Gradients of PercollProvide a Simple and Rapid Method That Consistently EnrichesNatural-Killer Cells. Journal of Immunological Methods 1988, 110 (2),161-168, and Childs, W. C.; Gibbons, R. J., Use of Percoll DensityGradients for Studying the Attachment of Bacteria to OralEpithelial-Cells. Journal of Dental Research 1988, 67 (5), 826-830, eachof which is incorporated by reference in its entirety. Using thisdensity-based method, libraries of transporters can be assayed andscreened for metal uptake efficiency given the direct physical change onyeast density, and better performing strains can be selected visuallyand filtered with increasingly more stringent density gradients (FIG.28A).

Expression of the metal transporter protein of interest can beincreased, its degradation in regular cellular pathways can be reduced,the transported metals can be shuttled into vacuoles for containment,and finally yeast tolerance can be improved to increased levels of metalaccumulation (FIG. 29A). One such transporter of interest is SMF1, apromiscuous divalent metal transporter which has been observed to uptakea range of metals such as manganese and cadmium. See Chen, X. Z.; Peng,J. B.; Cohen, A.; Nelson, H.; Nelson, N.; Hediger, M. A., Yeast Smf1Mediates H+-Coupled Iron Uptake with Concomitant Uncoupled CationCurrents. Journal of Biological Chemistry 1999, 274 (49), 35089-35094,which is incorporated by reference in its entirety. SMF1, unlike mostother metal transporter proteins, benefits from 30-40 years ofsequence-function research. As such, SMF1 mutants were rationallydesigned and compared for their metal uptake capacities. FIG. 28A showsincreased expression percentage as a function of SMF1 modifications,specifically conversion of lysine residues 33, 34 to arginine (denotedwith *) and a deletion of SMF1's degradation protein BSD2 (denotedABSD2). With these rationally designed modifications, cadmiumaccumulation was improved up to 4-fold greater than that of wild-typeyeast. Notably, the best performing mutant so far is capable ofexceeding 5 mg of cadmium per gram of yeast dry weight, which is beyondthe threshold for classifying plants as cadmium hyperaccumulators (FIG.29C). Modification of the SMF1 metal transporter confers metalhyperaccumulator status. See Rascio, N.; Navari-Izzo, F., Heavy MetalHyperaccumulating Plants: How and Why Do They Do It? And What Makes ThemSo Interesting? Plant Science 2011, 180 (2), 169-181, which isincorporated by reference in its entirety. Given the modularity of thisapproach, the selection can be tailored for other metals of interestsuch as radium and strontium, elements that are becoming increasinglyrecognized as radioactive contaminants since the Fukushima incident in2014. See Iwahana, Y.; Ohbuchi, A.; Koike, Y.; Kitano, M.; Nakamura, T.,Radioactive Nuclides in the Incinerator Ashes of Municipal Solid Wastesbefore and after the Accident at the Fukushima Nuclear Power Plant.Analytical Sciences 2013, 29 (1), 61-66, which is incorporated byreference in its entirety. A suite of yeast that can uptake a range oftoxic elements with high specificity can be developed in this way.

Another alternative strategy for metal remediation, in addition to yeastcell surface display and yeast capture and uptake, is to chemicallyreduce and precipitate metals from wastewaters. Chemical precipitationis the most widely used method for heavy metal remediation in industry,with hydrogen sulfide being one of the most commonly used chemicalprecipitants. See Fu, F. L.; Wang, Q., Removal of Heavy Metal Ions fromWastewaters: A Review. Journal of Environmental Management 2011, 92 (3),407-418, and Metcalf, E.; Eddy, H. P.; Tchobanoglous, G., WastewaterEngineering: Treatment, Disposal and Reuse. McGraw-Hill, New York 1991,each of which is incorporated by reference in its entirety. On atangential note, the wine industry discovered that yeasts are able toproduce an appreciable amount of hydrogen sulfide during fermentation,causing wine to smell and taste pungent. See Swiegers, J. H.; Pretorius,I. S., Modulation of Volatile Sulfur Compounds by Wine Yeast. AppliedMicrobiology and Biotechnology 2007, 74 (5), 954-960, which isincorporated by reference in its entirety. To inhibit sulfur productionfor better tasting wine, Linderholm et al. and Huang et al. discoveredthat knockouts of metabolic proteins, MET2, MET17 and CYS4, overproducehydrogen sulfide and are integral for complete sulfate metabolism forproduction of amino acids such as cysteine. See Linderholm, A. L.;Findleton, C. L.; Kumar, G.; Hong, Y.; Bisson, L. F., Identification ofGenes Affecting Hydrogen Sulfide Formation in Saccharomyces Cerevisiae.Applied and Environmental Microbiology 2008, 74 (5), 1418-1427, andHuang, C.; Roncoroni, M.; Gardner, R. C., MET2 Affects Production ofHydrogen Sulfide During Wine Fermentation. Applied Microbiology andBiotechnology 2014, 98 (16), 7125-7135, each of which is incorporated byreference in its entirety.

Because sulfur is a strong and reactive reducing and precipitating agentfor most transition metals (see Charerntanyarak, L., Heavy MetalsRemoval by Chemical Coagulation and Precipitation. Water Science andTechnology 1999, 39 (10-11), 135-138, which is incorporated by referencein its entirety), sulfur was aim to be overproduced, whereas the wineindustry has attempted to inhibit sulfur production (FIG. 30).Overproducing hydrogen sulfide serves as a powerful method for metalremediation in the form of chemical precipitation. From here, either thefirst or second bioremediation strategy can be employed to bind oruptake, respectively, these sulfide-metal complexes.

To utilize this strategy for bioremediation, the sulfur assimilatorycycle need to be interrupted at the point of sulfate to sulfideconversion (HSO₃ ⁻→S²⁻ [STOP]) and build up reactive sulfur in solution.Detection of hydrogen sulfide can be indirectly monitored from releasedhydrogen sulfide gas. Qualitative identification can be observed usinglead acetate strips while quantitative measurements can be performedusing sulfide detection columns, both of which undergo colorimetricchanges in the presence of sulfur (FIGS. 31A-31D). Monitoring colorchanges shows a production rate of 2 ppm/hr with a maximum production of55 pm during a complete culture cycle (<24 hr).

The successful mutant, ΔMET17, is able to produce roughly 2 ppm/hr ofhydrogen sulfide with a total of 55 ppm during a complete inoculationexperiment (>24 hr) in CSM. Cultures seeded with cadmium or copperprecipitate to CdS and CuS, respectively, showed a conversion of 90±5%of the initial metal concentration (FIGS. 34A-34B). Bioprecipitatedmetal sulfides, such as CdS, are regularly used as quantum dots. ReactedCdS from yeast behaves with characteristic excitation and emissionwavelengths of that of typical quantum dots. The size of the CdSparticles is approximately 50 nm in diameter, consistent with theexpected size that contributes to the quantum confinement of electronsgiven the distinct excitation and emission peaks (FIG. 32C).

These CdS particles are embedded in the cell wall using cross-sectioningTEM (FIG. 33A). The cell wall can then be digested using zymolase torelease the particles, and a uniform distribution of CdS particle sizes,as well as some CdS particles encapsulated in biologically derivedmaterial (e.g., protein or cell wall debris) were observed (FIG. 33B).

Packaging and Deployment

Another option is to store yeast in freeze-dried or active dry packages,much as baker's yeast are stored for consumer use, and distribute themfor on-demand applications. Large quantities of packaged yeast can bestored for later use during the events of disaster spills orcontamination leaks much like the BP oil spill, Fukushima nucleardisaster, and the Flint water crisis in 2010, 2012, and 2014,respectively. Deployable units of the engineered yeast can be createdfor on-site waste treatment. Options to create such a device may includeconstructing a filtering device that supports a resin-like bed of yeast.

Reducing the cost and scaling up the yeast technology can take advantageof the already established infrastructure for mass-producing yeast forconsumer purposes. The beer, wine, and pharmaceutical industries haveoptimized large-scale production of yeast, so there is already developedinfrastructure to produce yeast in mass. The production and consumptionof bread, beer, and medicine can be concurrently used to cleancontaminated waters (FIGS. 34A-34B).

Other embodiments are within the scope of the following claims.

What is claimed is:
 1. A composition for remediating a metal to treatwater comprising: a cell; and a first oligomer of a metal bindingprotein expressed on a surface of the cell via a linker; wherein thelinker is tethered to the first oligomer of the metal binding proteinand to a surface of the cell; wherein the metal binding protein hasspecificity for a metal; and wherein the first oligomer of the metalbinding protein expressed on the surface of the cell is capable ofaggregating with a second oligomer of the metal binding protein in thewater upon binding a metal, wherein the metal binding protein is fusedwith a high affinity protein including a metal binding domain, whereinthe metal binding domain has specificity for the metal.
 2. Thecomposition of claim 1, wherein the cell is yeast.
 3. The composition ofclaim 1, wherein the linker is tethered to the first oligomer of themetal binding protein.
 4. The composition of claim 1, wherein the secondoligomer of the metal binding protein is secreted from the cell.
 5. Thecomposition of claim 1, wherein the metal is a divalent metal.
 6. Thecomposition of claim 1, wherein the metal is a transition metal.
 7. Thecomposition of claim 1, wherein the metal is nickel, iron, copper,cobalt, lead, cadmium or mercury.
 8. The composition of claim 1, whereinthe metal binding protein is glutamine synthetase.
 9. The composition ofclaim 1, wherein the high affinity protein is a plant metallothionein.10. The composition of claim 2, wherein the metal binding protein isexpressed in a yeast host strain.
 11. A method of remediating a metal totreat water comprising: preparing a composition comprising: a cell; anda first oligomer of a metal binding protein expressed on a surface ofthe cell via a linker; wherein the linker is tethered to the firstoligomer of the metal binding protein and to a surface of the cell;wherein the metal binding protein has specificity for a metal; andwherein the first oligomer of the metal binding protein expressed on thesurface of the cell is capable of aggregating with a second oligomer ofthe metal binding protein in the water upon binding a metal, wherein themetal binding protein is fused with a high affinity protein including ametal binding domain, wherein the metal binding domain has specificityfor the metal; and contacting water with the composition.
 12. The methodof claim 11, further comprising adding the second oligomer of the metalbinding protein in the water.
 13. The method of claim 11, wherein thecell is yeast.
 14. The method of claim 11, wherein the linker is amonomer of the metal binding protein.
 15. The method n of claim 12,wherein the second oligomer of the metal binding protein is secretedfrom the cell.
 16. The method of claim 11, wherein the metal is adivalent metal.
 17. The method of claim 11, wherein the metal is nickel,iron, copper, cobalt, lead, cadmium or mercury.
 18. The method of claim11, wherein the metal binding protein is glutamine synthetase.
 19. Themethod of claim 11, wherein the metal binding protein is fused with ahigh affinity protein including a metal binding domain, wherein themetal binding domain has specificity for the metal.
 20. The method ofclaim 19, wherein the high affinity protein is a plant metallothionein.21. The method of claim 11, wherein the second oligomer of the metalbinding protein is secreted from the cell via a signal peptide.
 22. Themethod of claim 21, wherein the signal peptide is S. cerevisiae'sα-mating-factor.
 23. The method of claim 21, wherein the signal peptideis AGA1/2 or EXG1.
 24. The method of claim 11, wherein the method isperformed at 20° C. or lower temperature.
 25. The method of claim 11,wherein the metal binding protein is expressed in a yeast host strain.26. The method of claim 25, wherein the yeast host strain is PichiaPastoris.
 27. A method of making a composition for remediating a metalto treat water comprising: selecting a cell; expressing a metal bindingprotein on a surface of the cell; and tethering a first oligomer of ametal binding protein expressed on a surface of the cell via a linker;wherein the linker is tethered to the first oligomer of the metalbinding protein and to a surface of the cell; wherein the metal bindingprotein has specificity for a metal; and wherein the first oligomer ofthe metal binding protein expressed on the surface of the cell iscapable of aggregating with a second oligomer of the metal bindingprotein in the water upon binding a metal, wherein the metal bindingprotein is fused with a high affinity protein including a metal bindingdomain, wherein the metal binding domain has specificity for the metal.28. A method of mining a metal comprising: preparing a compositioncomprising: a cell; and a first oligomer of a metal binding proteinexpressed on a surface of the cell via a linker; wherein the linker istethered to the first oligomer of the metal binding protein and to asurface of the cell; wherein the metal binding protein has specificityfor a metal; and wherein the first oligomer of the metal binding proteinexpressed on the surface of the cell is capable of aggregating with asecond oligomer of the metal binding protein in the water upon binding ametal, wherein the metal binding protein is fused with a high affinityprotein including a metal binding domain, wherein the metal bindingdomain has specificity for the metal; contacting water with thecomposition; and lysing the cell to obtain the metal.
 29. The method ofclaim 28, wherein the metal is a noble metal.
 30. The method of claim29, wherein the noble metal is gold, silver or platinum.
 31. The methodof claim 28, wherein the metal is a rare-earth metal.
 32. The method ofclaim 31, wherein the rare-earth metal is cerium (Ce), dysprosium (Dy),erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho), lanthanum(La), lutetium (Lu), neodymium (Nd), praseodymium (Pr), promethium (Pm),samarium (Sm), scandium (Sc), terbium (Tb), thulium (Tm), ytterbium (Yb)or yttrium (Y).
 33. The composition of claim 1, wherein the linker isattached to the surface of the cell.